{"gene":"RAMP3","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2001,"finding":"Seven amino acid sequences in the extracellular N-terminal domain of RAMP3 (residues 59-65) are critical for high-affinity adrenomedullin binding and AM-evoked cAMP production when co-expressed with CRLR in HEK293 cells; deletion of these residues significantly attenuated 125I-AM binding and AM-evoked cAMP production despite full cell surface expression of the receptor heterodimer.","method":"Deletion mutagenesis, 125I-AM binding assay, cAMP production assay in HEK293 cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro mutagenesis with both ligand binding and functional (cAMP) readouts in a single study","pmids":["11591721"],"is_preprint":false},{"year":2003,"finding":"N-glycosylation and conserved cysteine residues in RAMP3 are critical for functional expression of the CRLR/RAMP3 adrenomedullin receptor: elimination of all N-glycans significantly inhibited 125I-AM binding and increased EC50 for AM; mutation of all six cysteines to serines abolished AM binding and receptor function, indicating each cysteine participates in disulfide bond formation.","method":"Xenopus oocyte expression system, 125I-AM binding assay, site-directed mutagenesis of N-glycosylation sites and cysteines","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution in oocyte system with mutagenesis and functional readouts (binding + cAMP), multiple orthogonal approaches","pmids":["12939163"],"is_preprint":false},{"year":2003,"finding":"CL/RAMP3 and CL/RAMP2 form pharmacologically distinct adrenomedullin receptors: AM22-52 selectively antagonizes CL/RAMP2 over CL/RAMP3 in a species-dependent manner, while CGRP8-37 shows opposite selectivity, demonstrating that RAMP identity determines antagonist pharmacology of the receptor complex.","method":"Radioligand binding and functional antagonism assays in COS7 cells (transient expression) and endogenously expressing cell lines (Rat2, L6)","journal":"British journal of pharmacology","confidence":"High","confidence_rationale":"Tier 2 / Strong — pharmacological characterization across multiple species combinations and cell lines, replicated across endogenous and overexpression systems","pmids":["12970090"],"is_preprint":false},{"year":2010,"finding":"RAMP1-RAMP3 chimera analysis revealed that helix 1 and residues 62-69 in helix 2 of RAMP1 are critical for CLR trafficking (a marker of CLR association), and modeling indicates these exchanges alter the CLR recognition site; residues 86-89 at the helix 2-3 junction affect peptide potency but not CLR trafficking, suggesting a role in ligand binding pocket formation.","method":"RAMP1/RAMP3 chimera expression in cells, CLR trafficking assay, agonist potency assay, molecular modeling","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chimera/mutagenesis with functional readouts, supported by modeling, single lab","pmids":["20017504"],"is_preprint":false},{"year":2011,"finding":"Glu74 of RAMP3 contributes to AM binding and potency at the CLR/RAMP3 (AM2) receptor; substitution with Phe reduced AM potency, while Trp, Tyr, Ala, Ser, Thr, Arg, and Asn substitutions had mostly small effects, indicating the geometry and charge at position 74 contribute to AM interaction specificity.","method":"Site-directed mutagenesis of RAMP3, co-expression with CLR in COS7 cells, cAMP assay, cell surface ELISA","journal":"Peptides","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — mutagenesis with functional assay, single lab, single study","pmids":["21402116"],"is_preprint":false},{"year":2004,"finding":"CRLR/RAMP2 and CRLR/RAMP3 receptors mediate adrenomedullin-induced HUVEC migration, invasion, and differentiation into cord-like structures on Matrigel; blocking these receptors inhibited AM-promoted angiogenic effects on endothelial cells in vitro.","method":"Migration/invasion assays with receptor-blocking approaches in HUVECs, Matrigel tube formation assay","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional angiogenesis assays with receptor-specific antagonism, single lab","pmids":["14712479"],"is_preprint":false},{"year":2019,"finding":"RAMP3 associates with atypical chemokine receptor 3 (ACKR3) and is required for rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following AM or SDF-1/CXCL12 binding, without changing G-protein coupling; this recycling enables formation of dynamic spatiotemporal chemotactic gradients, and genetic deletion of RAMP3 in mice abolishes directed cell migration during retinal angiogenesis.","method":"BRET-based interaction assay, cell-surface expression assay, Rab4-vesicle trafficking analysis, RAMP3 knockout mouse retinal angiogenesis model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (BRET, trafficking, KO mouse phenotype), mechanistic detail on Rab4 recycling pathway","pmids":["31712427"],"is_preprint":false},{"year":2014,"finding":"RAMP3 knockout mice develop normally without major vascular abnormalities, but display delayed drainage of subcutaneous lymphatic vessels (shown by ICG lymphangiography), delayed intestinal chyle transport, more severe interstitial edema in a tail lymphedema model, and impaired migration of isolated lymphatic endothelial cells in scratch-wound assays; AM administration failed to rescue re-endothelialization in RAMP3-/- cells.","method":"RAMP3 knockout mouse generation, ICG lymphangiography, tail lymphedema model, scratch-wound assay with isolated lymphatic endothelial cells","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic KO with multiple in vivo and in vitro functional readouts, mechanistic link to AM-RAMP3 signaling in lymphatic biology","pmids":["25264174"],"is_preprint":false},{"year":2021,"finding":"In a pressure-overload (TAC) model, RAMP3 knockout mice show reduced systolic function and enhanced fibrosis appearing after 4 weeks (later than RAMP2-deficient mice), with a characteristic reduction in cardiac lymphatic vessels, indicating the AM-RAMP3 system is required for later cardiac adaptation to stress through regulation of lymphatic vessels.","method":"Cardiomyocyte-specific and global RAMP3 knockout mice subjected to transverse aortic constriction, echocardiography, histological fibrosis assessment, cardiac lymphatic vessel analysis","journal":"Endocrinology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with multiple in vivo cardiac functional readouts, mechanistic differentiation from RAMP2 pathway in same study","pmids":["33545715"],"is_preprint":false},{"year":2019,"finding":"RAMP3 deficiency in mice suppresses tumor metastasis by reducing podoplanin (PDPN)-positive cancer-associated fibroblasts (CAFs) at metastatic sites; RAMP3 was shown to mediate Src/Cas/PDPN signaling, and RAMP3-deficient CAFs displayed suppressed migration, proliferation, and metastasis in co-culture with tumor cells.","method":"RAMP3 knockout mice with splenic injection pancreatic cancer model, immunostaining for PDPN+ CAFs, in vitro co-culture assays, signaling pathway analysis (Src/Cas/PDPN)","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse model with in vivo metastasis readout and mechanistic in vitro follow-up, single lab","pmids":["31754214"],"is_preprint":false},{"year":2005,"finding":"PTH induces RAMP3 mRNA expression as a primary response gene in primary mouse osteoblasts primarily through the cAMP-PKA signaling pathway; this was demonstrated by showing that cycloheximide does not block PTH-induced RAMP3 expression (primary response), forskolin mimics the effect, and the PKA inhibitor H89 blocks both PTH- and forskolin-induced RAMP3 upregulation.","method":"RT-PCR in primary mouse osteoblasts, pharmacological dissection with PKA inhibitor (H89), PKC activator (PMA), calcium ionophore (ionomycin), and protein synthesis inhibitor (cycloheximide)","journal":"Calcified tissue international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple pharmacological tools dissecting signaling pathway, primary cell system, single lab","pmids":["16075364"],"is_preprint":false},{"year":2015,"finding":"RAMP3 associates with CALCRL (CRLR) in vascular smooth muscle cells to mediate ADM-induced cAMP production; knockdown of RAMP3 (but not RAMP2) inhibited ADM-induced cAMP production, and proximity ligation assay showed ADM increases CALCRL-RAMP3 association; RAMP3 knockdown inhibited ADM interaction with CALCRL.","method":"siRNA knockdown of RAMP2 or RAMP3 in mesenteric artery VSMCs, cAMP assay, proximity ligation assay","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown plus proximity ligation assay for direct protein interaction, functional readout, single lab","pmids":["26423127"],"is_preprint":false},{"year":2019,"finding":"In AMY3 receptors (CTR/RAMP3 heterodimers), RAMP3 fundamentally alters the dynamic role of CTR extracellular loops 2 and 3 in signal propagation compared to CTR alone; alanine scanning mutagenesis of ECL2 and ECL3 showed distinct peptide-specific effects on cAMP accumulation and ERK phosphorylation, demonstrating RAMP3 changes the signaling landscape of CTR in a peptide-dependent manner.","method":"Alanine scanning mutagenesis of CTR ECL2 and ECL3 in the context of RAMP3 co-expression, cAMP accumulation assay, ERK phosphorylation assay","journal":"ACS pharmacology & translational science","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — systematic mutagenesis with two orthogonal signaling readouts, single lab","pmids":["32219220"],"is_preprint":false},{"year":2009,"finding":"RAMP3 is an early transcriptional target gene of the canonical Wnt pathway; stimulation of C57MG cells with Wnt-3a induced RAMP3 mRNA expression between 1 and 6 hours post-stimulation.","method":"Wnt-3a stimulation of C57MG cells, temporal mRNA expression analysis","journal":"Cell biology international","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method (RT-PCR), no mechanistic follow-up on RAMP3 protein function","pmids":["19353769"],"is_preprint":false},{"year":2019,"finding":"RAMP3 knockout mice on high-fat diet show glucose intolerance and altered amylin anorectic responses; RAMP3 KO mice respond to salmon calcitonin (CTR agonist) but not amylin for food intake suppression, establishing RAMP3 as necessary for amylin's anorectic effect and glucose homeostasis mediated through the CTR/RAMP3 (AMY3) receptor.","method":"RAMP3 global KO mice, high-fat diet feeding, glucose tolerance tests, food intake pharmacology with amylin and salmon calcitonin, c-Fos immunostaining in area postrema","journal":"Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with multiple metabolic and pharmacological readouts, single lab","pmids":["31881259"],"is_preprint":false},{"year":2018,"finding":"RAMP3 deficiency in ovariectomized (postmenopausal) mice exacerbates obesity, adipose tissue weight gain, insulin resistance, and hepatic steatosis; GPR30 (membrane estrogen receptor) expression was downregulated in periuterine WAT from RAMP3-/- OVX mice, suggesting a GPR30-RAMP3 interaction in metabolic regulation.","method":"RAMP3 KO mice subjected to ovariectomy plus high-fat diet, metabolic phenotyping, adipose gene expression analysis","journal":"Peptides","confidence":"Low","confidence_rationale":"Tier 3 / Weak — KO phenotype with expression data suggesting GPR30-RAMP3 link, no direct biochemical demonstration of interaction","pmids":["30385288"],"is_preprint":false},{"year":2024,"finding":"RAMP3 binds to atypical chemokine receptor 2 (ACKR2) in HCC tumor cells and promotes membrane distribution of ACKR2 through RAB4-positive vesicles, thereby facilitating CCL2 scavenging by ACKR2 and inhibiting STAT3 and AKT phosphorylation; RAMP3 also suppressed CD11b+/Gr-1+ myeloid cell infiltration and neovascularization in tumors through ACKR2.","method":"Co-IP/protein-protein interaction assays in HCC cell lines (Huh7, HepG2, Hepa1-6), RAB4 vesicle trafficking assay, syngeneic mouse tumor model, CCL2 measurement, STAT3/AKT phosphorylation assay","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding assay combined with in vivo syngeneic tumor model and signaling readouts, mechanistically consistent with RAMP3-ACKR3 interaction previously described","pmids":["39437486"],"is_preprint":false},{"year":2025,"finding":"RAMP3 interacts with GLP-1R and biases its signaling toward Ca2+ mobilization away from canonical cAMP-driven signaling; RAMP3 reduces Gαs activation but increases secondary coupling to Gαq and Gαi, leading to enhanced glucose-stimulated insulin secretion in cells overexpressing RAMP3; RAMP3 KO mice show reduced sensitivity to GLP-1 in glucose and insulin tolerance tests.","method":"BRET interaction assay, G protein coupling assays, Ca2+ mobilization assay, cAMP assay, glucose-stimulated insulin secretion in cells, RAMP3 KO mouse glucose/insulin tolerance tests","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal signaling assays plus KO mouse phenotype, preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.01.24.634724"],"is_preprint":true},{"year":2024,"finding":"The AMY3R (CTR/RAMP3) has a stronger transmembrane domain interface than AMY1R/AMY2R, yielding a more stable heterodimer; human and salmon calcitonin agonists promote AMY3R dissociation whereas amylin promotes AMY1/2R subunit association; these distinct subunit equilibriums modulated by agonists determine cAMP signaling output.","method":"Novel biochemical assay resolving AMYR heterodimers and free subunits, live cell membrane assays, G protein coupling assays, cAMP signaling assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — novel biochemical reconstitution-type assay with multiple signaling readouts, preprint not yet peer-reviewed","pmids":["bio_10.1101_2024.10.09.617487"],"is_preprint":true}],"current_model":"RAMP3 is a single-transmembrane accessory protein that functions as a molecular chaperone and allosteric modulator of multiple GPCRs: it heterodimerizes with CLR (CRLR) to form the AM2 adrenomedullin receptor (with N-glycosylation and six cysteine disulfide bonds in its extracellular domain critical for function), associates with CTR to form the AMY3 amylin receptor (with RAMP3 altering ECL2/ECL3 signal propagation and stabilizing the heterodimer), interacts with ACKR3 and ACKR2 to promote Rab4-dependent rapid receptor recycling to the plasma membrane (enabling chemotactic gradient formation), and interacts with GLP-1R to bias signaling toward Gαq/Gαi and Ca2+ mobilization enhancing insulin secretion; in vivo, RAMP3 is specifically required for lymphatic vessel drainage, later cardiac adaptation to pressure overload, directed retinal angiogenesis, and the anorectic/glucose homeostatic effects of amylin."},"narrative":{"mechanistic_narrative":"RAMP3 is a single-transmembrane accessory protein that determines the pharmacology, trafficking, and signaling output of multiple G protein-coupled receptors by heterodimerizing with them at the cell surface [PMID:11591721, PMID:12970090, PMID:31712427]. Its extracellular N-terminal domain forms the ligand interface of the CLR/RAMP3 (AM2) adrenomedullin receptor: residues 59-65 and Glu74 are critical for high-affinity adrenomedullin binding and AM-evoked cAMP production, and N-glycosylation together with six cysteine-stabilized disulfide bonds are required for functional receptor expression [PMID:11591721, PMID:12939163, PMID:21402116]. RAMP3 identity dictates the antagonist selectivity of the receptor complex, distinguishing CLR/RAMP3 from CLR/RAMP2 pharmacologically [PMID:12970090], and helix 1 and helix 2 residues govern CLR association and trafficking versus ligand-pocket formation [PMID:20017504]. Beyond CLR, RAMP3 partners with the calcitonin receptor (CTR) to form the AMY3 amylin receptor, where it reshapes the contribution of CTR extracellular loops 2 and 3 to signal propagation and stabilizes a transmembrane-domain heterodimer interface whose agonist-driven dissociation tunes cAMP output [PMID:32219220, PMID:bio_10.1101_2024.10.09.617487]. RAMP3 also acts as a recycling chaperone for the atypical chemokine receptors ACKR3 and ACKR2, promoting their Rab4-dependent return to the plasma membrane to sustain chemokine scavenging and chemotactic gradient formation [PMID:31712427, PMID:39437486], and biases GLP-1R signaling away from Gαs/cAMP toward Gαq/Gαi and Ca2+ mobilization to enhance glucose-stimulated insulin secretion [PMID:bio_10.1101_2025.01.24.634724]. In vivo, RAMP3 is dispensable for baseline vascular development but is specifically required for lymphatic vessel drainage, directed retinal angiogenesis, later cardiac adaptation to pressure overload through cardiac lymphatics, and the anorectic and glucose-homeostatic effects of amylin [PMID:31712427, PMID:25264174, PMID:33545715, PMID:31881259].","teleology":[{"year":2001,"claim":"Established which part of RAMP3 contacts ligand, answering how an accessory protein contributes to adrenomedullin pharmacology rather than merely escorting the receptor.","evidence":"Deletion mutagenesis with 125I-AM binding and cAMP readouts in HEK293 cells co-expressing CRLR","pmids":["11591721"],"confidence":"High","gaps":["Does not resolve the structural fold of the bound complex","No information on RAMP3 partners beyond CLR"]},{"year":2003,"claim":"Defined the structural requirements (N-glycosylation, disulfide bonds) for a functional CLR/RAMP3 receptor and showed RAMP identity dictates antagonist pharmacology.","evidence":"Oocyte and COS7 reconstitution with cysteine/glycosylation mutagenesis and cross-species pharmacological antagonism","pmids":["12939163","12970090"],"confidence":"High","gaps":["Disulfide connectivity not directly mapped","Species-dependence of antagonist selectivity not mechanistically explained"]},{"year":2005,"claim":"Showed RAMP3 expression is regulated, identifying it as a cAMP-PKA-driven primary response gene downstream of PTH in osteoblasts.","evidence":"RT-PCR with cycloheximide, forskolin, and H89 pharmacological dissection in primary mouse osteoblasts","pmids":["16075364"],"confidence":"Medium","gaps":["Transcription factors mediating induction not identified","Functional consequence of RAMP3 induction in bone not tested"]},{"year":2010,"claim":"Separated the RAMP determinants of receptor association/trafficking from those shaping the ligand-binding pocket, refining how RAMP3-type proteins remodel CLR.","evidence":"RAMP1/RAMP3 chimera analysis with CLR trafficking and agonist potency assays plus molecular modeling","pmids":["20017504"],"confidence":"Medium","gaps":["Modeling-based, no experimental structure","Single lab"]},{"year":2011,"claim":"Pinpointed a single residue (Glu74) contributing charge/geometry to adrenomedullin specificity at the AM2 receptor.","evidence":"Site-directed mutagenesis of RAMP3 with cAMP and surface-ELISA readouts in COS7 cells","pmids":["21402116"],"confidence":"Medium","gaps":["Single residue context only","No structural confirmation of the contact"]},{"year":2014,"claim":"Moved RAMP3 from in vitro pharmacology to physiology, showing it is selectively required for lymphatic vessel function rather than general vascular development.","evidence":"RAMP3 knockout mice with ICG lymphangiography, tail lymphedema model, and lymphatic endothelial scratch-wound assays","pmids":["25264174"],"confidence":"High","gaps":["Receptor partner mediating the lymphatic phenotype not isolated in vivo","Cell-autonomous versus systemic contributions not separated"]},{"year":2019,"claim":"Revealed a chaperone/recycling function for RAMP3 distinct from G-protein modulation, explaining how it controls chemokine receptor surface levels and gradient formation.","evidence":"BRET interaction, Rab4-vesicle trafficking analysis, and RAMP3 KO mouse retinal angiogenesis","pmids":["31712427"],"confidence":"High","gaps":["Recruitment mechanism of RAMP3 to Rab4 vesicles unknown","Generality of the recycling role across other GPCRs untested at the time"]},{"year":2019,"claim":"Extended RAMP3 function to AMY3 (CTR/RAMP3) signaling and to tumor-stromal biology, showing peptide-specific reshaping of CTR extracellular-loop signaling and a metastasis-promoting role via CAFs.","evidence":"Alanine scanning of CTR ECL2/ECL3 with cAMP and ERK readouts; RAMP3 KO mouse metastasis model with Src/Cas/PDPN signaling analysis","pmids":["32219220","31754214"],"confidence":"Medium","gaps":["Direct structural basis of ECL remodeling not resolved","Mechanistic link between RAMP3 GPCR activity and Src/Cas/PDPN in CAFs not fully traced"]},{"year":2021,"claim":"Linked RAMP3 to organ-level stress adaptation, showing its loss impairs late cardiac compensation to pressure overload via reduced cardiac lymphatics.","evidence":"Global and cardiomyocyte-specific RAMP3 KO mice with transverse aortic constriction, echocardiography, fibrosis histology, and lymphatic vessel analysis","pmids":["33545715"],"confidence":"High","gaps":["Temporal divergence from RAMP2 pathway mechanistically unexplained","Direct ligand-receptor axis in cardiac lymphatics not defined"]},{"year":2019,"claim":"Established RAMP3 as physiologically required for amylin's anorectic and glucose-homeostatic actions through the AMY3 receptor.","evidence":"RAMP3 global KO mice on high-fat diet with glucose tolerance tests, amylin/salmon calcitonin food-intake pharmacology, and area postrema c-Fos staining","pmids":["31881259"],"confidence":"Medium","gaps":["Central versus peripheral site of action not fully resolved","Single lab"]},{"year":2024,"claim":"Generalized the Rab4-recycling chaperone mechanism to ACKR2, connecting RAMP3 to CCL2 scavenging and an anti-tumor immune/vascular phenotype.","evidence":"Co-IP, RAB4 trafficking, syngeneic HCC tumor model, CCL2 measurement, and STAT3/AKT phosphorylation assays","pmids":["39437486"],"confidence":"Medium","gaps":["Direct binding interface with ACKR2 not mapped","Reconciliation with metastasis-promoting role in other models unresolved"]},{"year":2025,"claim":"Showed RAMP3 acts as a signaling-bias modulator of GLP-1R, redirecting it from Gαs/cAMP toward Gαq/Gαi and Ca2+ to enhance insulin secretion.","evidence":"BRET interaction, G protein coupling and Ca2+/cAMP assays, glucose-stimulated insulin secretion, and RAMP3 KO mouse tolerance tests (preprint)","pmids":["bio_10.1101_2025.01.24.634724"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Structural basis of biased coupling not determined"]},{"year":2024,"claim":"Provided a biophysical basis for AMY3 receptor behavior, showing a stronger TM interface and agonist-dependent subunit equilibrium that tunes signaling.","evidence":"Novel heterodimer-resolving biochemical assay with live-cell membrane and G protein/cAMP readouts (preprint)","pmids":["bio_10.1101_2024.10.09.617487"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","In vivo relevance of the dissociation equilibrium untested"]},{"year":null,"claim":"How RAMP3 selects among its many GPCR partners in a given cell, and whether its recycling-chaperone versus pharmacology-modulating roles are coordinated, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of RAMP3 in complex with non-CLR partners reported in the corpus","Determinants of partner choice in native tissues unknown","Mechanism coupling RAMP3 expression regulation to its receptor functions undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2,12,17]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[6,16,17]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,18]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,6,16]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[6,16]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,6,12,17]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[6,16]}],"complexes":["CLR/RAMP3 (AM2) receptor","CTR/RAMP3 (AMY3) amylin receptor"],"partners":["CALCRL","CALCR","ACKR3","ACKR2","GLP1R"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O60896","full_name":"Receptor activity-modifying protein 3","aliases":["Calcitonin-receptor-like receptor activity-modifying protein 3","CRLR activity-modifying protein 3"],"length_aa":148,"mass_kda":16.5,"function":"Accessory protein that interacts with and modulates the function of G-protein coupled receptors including calcitonin gene-related peptide type 1 receptor (CALCRL), calcitonin receptor (CALCR) and G-protein coupled estrogen receptor 1 (GPER1) (PubMed:23674134, PubMed:9620797). Required for the transport of CALCRL and GPER1 receptors to the plasma membrane (PubMed:23674134, PubMed:9620797). Plays a role in cardioprotection by reducing cardiac hypertrophy and perivascular fibrosis in a GPER1-dependent manner (PubMed:23674134). Together with CALCRL, form a receptor complex for adrenomedullin/ADM and intermedin/ADM2 (PubMed:32296767). Together with CALCR, act as a receptor complex for amylin/IAPP (PubMed:35324283)","subcellular_location":"Cell membrane; Membrane","url":"https://www.uniprot.org/uniprotkb/O60896/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RAMP3","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/RAMP3","total_profiled":1310},"omim":[{"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"},{"mim_id":"603273","title":"TUMOR PROTEIN p63; TP63","url":"https://www.omim.org/entry/603273"},{"mim_id":"601990","title":"TUMOR PROTEIN p73; TP73","url":"https://www.omim.org/entry/601990"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RAMP3"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"O60896","domains":[{"cath_id":"1.10.150.510","chopping":"27-102","consensus_level":"high","plddt":96.1451,"start":27,"end":102}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O60896","model_url":"https://alphafold.ebi.ac.uk/files/AF-O60896-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O60896-F1-predicted_aligned_error_v6.png","plddt_mean":87.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RAMP3","jax_strain_url":"https://www.jax.org/strain/search?query=RAMP3"},"sequence":{"accession":"O60896","fasta_url":"https://rest.uniprot.org/uniprotkb/O60896.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O60896/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O60896"}},"corpus_meta":[{"pmid":"12970090","id":"PMC_12970090","title":"CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors: a comparison of effects of adrenomedullin22-52, CGRP8-37 and BIBN4096BS.","date":"2003","source":"British journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/12970090","citation_count":117,"is_preprint":false},{"pmid":"14712479","id":"PMC_14712479","title":"Effects of adrenomedullin on endothelial cells in the multistep process of angiogenesis: involvement of CRLR/RAMP2 and CRLR/RAMP3 receptors.","date":"2004","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/14712479","citation_count":111,"is_preprint":false},{"pmid":"12051717","id":"PMC_12051717","title":"Increased myocardial expression of RAMP1 and RAMP3 in rats with chronic heart failure.","date":"2002","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/12051717","citation_count":55,"is_preprint":false},{"pmid":"11591721","id":"PMC_11591721","title":"The seven amino acids of human RAMP2 (86) and RAMP3 (59) are critical for agonist binding to human adrenomedullin receptors.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11591721","citation_count":53,"is_preprint":false},{"pmid":"19353769","id":"PMC_19353769","title":"Chronological expression of Wnt target genes Ccnd1, Myc, Cdkn1a, Tfrc, Plf1 and Ramp3.","date":"2009","source":"Cell biology international","url":"https://pubmed.ncbi.nlm.nih.gov/19353769","citation_count":46,"is_preprint":false},{"pmid":"31712427","id":"PMC_31712427","title":"RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis.","date":"2019","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/31712427","citation_count":44,"is_preprint":false},{"pmid":"12801991","id":"PMC_12801991","title":"Changes in the expression of calcitonin receptor-like receptor, receptor activity-modifying protein (RAMP) 1, RAMP2, and RAMP3 in rat uterus during pregnancy, labor, and by steroid hormone treatments.","date":"2003","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/12801991","citation_count":41,"is_preprint":false},{"pmid":"31881259","id":"PMC_31881259","title":"RAMP1 and RAMP3 Differentially Control Amylin's Effects on Food Intake, Glucose and Energy Balance in Male and Female Mice.","date":"2019","source":"Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/31881259","citation_count":36,"is_preprint":false},{"pmid":"25264174","id":"PMC_25264174","title":"Functional differentiation of RAMP2 and RAMP3 in their regulation of the vascular system.","date":"2014","source":"Journal of molecular and cellular cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/25264174","citation_count":34,"is_preprint":false},{"pmid":"12939163","id":"PMC_12939163","title":"N-Glycosylation and conserved cysteine residues in RAMP3 play a critical role for the functional expression of CRLR/RAMP3 adrenomedullin receptor.","date":"2003","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12939163","citation_count":23,"is_preprint":false},{"pmid":"31754214","id":"PMC_31754214","title":"Deficiency of the adrenomedullin-RAMP3 system suppresses metastasis through the modification of cancer-associated fibroblasts.","date":"2019","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/31754214","citation_count":22,"is_preprint":false},{"pmid":"21402116","id":"PMC_21402116","title":"Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors.","date":"2011","source":"Peptides","url":"https://pubmed.ncbi.nlm.nih.gov/21402116","citation_count":22,"is_preprint":false},{"pmid":"15623431","id":"PMC_15623431","title":"RAMP2 and RAMP3 mRNA levels are increased in failing rat cardiomyocytes and associated with increased responsiveness to adrenomedullin.","date":"2004","source":"Journal of molecular and cellular cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/15623431","citation_count":19,"is_preprint":false},{"pmid":"16075364","id":"PMC_16075364","title":"Parathyroid hormone induces receptor activity modifying protein-3 (RAMP3) expression primarily via 3',5'-cyclic adenosine monophosphate signaling in osteoblasts.","date":"2005","source":"Calcified tissue international","url":"https://pubmed.ncbi.nlm.nih.gov/16075364","citation_count":19,"is_preprint":false},{"pmid":"32219220","id":"PMC_32219220","title":"Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor: RAMP3 Alters Signal Propagation through Extracellular Loops of the Calcitonin Receptor.","date":"2019","source":"ACS pharmacology & translational science","url":"https://pubmed.ncbi.nlm.nih.gov/32219220","citation_count":11,"is_preprint":false},{"pmid":"30385288","id":"PMC_30385288","title":"RAMP3 deficiency enhances postmenopausal obesity and metabolic disorders.","date":"2018","source":"Peptides","url":"https://pubmed.ncbi.nlm.nih.gov/30385288","citation_count":10,"is_preprint":false},{"pmid":"33545715","id":"PMC_33545715","title":"Adrenomedullin-RAMP2 and -RAMP3 Systems Regulate Cardiac Homeostasis during Cardiovascular Stress.","date":"2021","source":"Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/33545715","citation_count":9,"is_preprint":false},{"pmid":"26423127","id":"PMC_26423127","title":"Involvement of Receptor Activity-Modifying Protein 3 (RAMP3) in the Vascular Actions of Adrenomedullin in Rat Mesenteric Artery Smooth Muscle Cells.","date":"2015","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/26423127","citation_count":8,"is_preprint":false},{"pmid":"20017504","id":"PMC_20017504","title":"Structure-function analysis of RAMP1-RAMP3 chimeras.","date":"2010","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20017504","citation_count":6,"is_preprint":false},{"pmid":"16376586","id":"PMC_16376586","title":"Induction of BAALC and down regulation of RAMP3 in astrocytes treated with differentiation inducers.","date":"2005","source":"Cell biology international","url":"https://pubmed.ncbi.nlm.nih.gov/16376586","citation_count":6,"is_preprint":false},{"pmid":"32550545","id":"PMC_32550545","title":"Analysis of RAMP3 gene polymorphism with body composition and bone density in young and elderly women.","date":"2019","source":"Gene: X","url":"https://pubmed.ncbi.nlm.nih.gov/32550545","citation_count":5,"is_preprint":false},{"pmid":"33711314","id":"PMC_33711314","title":"Adrenomedullin 2 and 5 activate the calcitonin receptor-like receptor (clr) - Receptor activity-modifying protein 3 (ramp3) receptor complex in Xenopus tropicalis.","date":"2021","source":"General and comparative endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/33711314","citation_count":3,"is_preprint":false},{"pmid":"34530989","id":"PMC_34530989","title":"Analysis of RAMP3 gene polymorphism with body composition and bone density in young and elderly women.","date":"2019","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/34530989","citation_count":2,"is_preprint":false},{"pmid":"39437486","id":"PMC_39437486","title":"-RAMP3 promotes hepatocellular carcinoma tumor cell-mediated CCL2 degradation by supporting membrane distribution of ACKR2.","date":"2024","source":"International immunopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/39437486","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.01.24.634724","title":"Receptor Activity-Modifying Protein 3 enhances GLP-1-mediated Insulin Secretion","date":"2025-01-27","source":"bioRxiv","url":"https://doi.org/10.1101/2025.01.24.634724","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}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16961,"output_tokens":5050,"usd":0.063316,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13351,"output_tokens":4256,"usd":0.086578,"stage2_stop_reason":"end_turn"},"total_usd":0.149894,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"Seven amino acid sequences in the extracellular N-terminal domain of RAMP3 (residues 59-65) are critical for high-affinity adrenomedullin binding and AM-evoked cAMP production when co-expressed with CRLR in HEK293 cells; deletion of these residues significantly attenuated 125I-AM binding and AM-evoked cAMP production despite full cell surface expression of the receptor heterodimer.\",\n      \"method\": \"Deletion mutagenesis, 125I-AM binding assay, cAMP production assay in HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro mutagenesis with both ligand binding and functional (cAMP) readouts in a single study\",\n      \"pmids\": [\"11591721\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"N-glycosylation and conserved cysteine residues in RAMP3 are critical for functional expression of the CRLR/RAMP3 adrenomedullin receptor: elimination of all N-glycans significantly inhibited 125I-AM binding and increased EC50 for AM; mutation of all six cysteines to serines abolished AM binding and receptor function, indicating each cysteine participates in disulfide bond formation.\",\n      \"method\": \"Xenopus oocyte expression system, 125I-AM binding assay, site-directed mutagenesis of N-glycosylation sites and cysteines\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution in oocyte system with mutagenesis and functional readouts (binding + cAMP), multiple orthogonal approaches\",\n      \"pmids\": [\"12939163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CL/RAMP3 and CL/RAMP2 form pharmacologically distinct adrenomedullin receptors: AM22-52 selectively antagonizes CL/RAMP2 over CL/RAMP3 in a species-dependent manner, while CGRP8-37 shows opposite selectivity, demonstrating that RAMP identity determines antagonist pharmacology of the receptor complex.\",\n      \"method\": \"Radioligand binding and functional antagonism assays in COS7 cells (transient expression) and endogenously expressing cell lines (Rat2, L6)\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — pharmacological characterization across multiple species combinations and cell lines, replicated across endogenous and overexpression systems\",\n      \"pmids\": [\"12970090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RAMP1-RAMP3 chimera analysis revealed that helix 1 and residues 62-69 in helix 2 of RAMP1 are critical for CLR trafficking (a marker of CLR association), and modeling indicates these exchanges alter the CLR recognition site; residues 86-89 at the helix 2-3 junction affect peptide potency but not CLR trafficking, suggesting a role in ligand binding pocket formation.\",\n      \"method\": \"RAMP1/RAMP3 chimera expression in cells, CLR trafficking assay, agonist potency assay, molecular modeling\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chimera/mutagenesis with functional readouts, supported by modeling, single lab\",\n      \"pmids\": [\"20017504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Glu74 of RAMP3 contributes to AM binding and potency at the CLR/RAMP3 (AM2) receptor; substitution with Phe reduced AM potency, while Trp, Tyr, Ala, Ser, Thr, Arg, and Asn substitutions had mostly small effects, indicating the geometry and charge at position 74 contribute to AM interaction specificity.\",\n      \"method\": \"Site-directed mutagenesis of RAMP3, co-expression with CLR in COS7 cells, cAMP assay, cell surface ELISA\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — mutagenesis with functional assay, single lab, single study\",\n      \"pmids\": [\"21402116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CRLR/RAMP2 and CRLR/RAMP3 receptors mediate adrenomedullin-induced HUVEC migration, invasion, and differentiation into cord-like structures on Matrigel; blocking these receptors inhibited AM-promoted angiogenic effects on endothelial cells in vitro.\",\n      \"method\": \"Migration/invasion assays with receptor-blocking approaches in HUVECs, Matrigel tube formation assay\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional angiogenesis assays with receptor-specific antagonism, single lab\",\n      \"pmids\": [\"14712479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RAMP3 associates with atypical chemokine receptor 3 (ACKR3) and is required for rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following AM or SDF-1/CXCL12 binding, without changing G-protein coupling; this recycling enables formation of dynamic spatiotemporal chemotactic gradients, and genetic deletion of RAMP3 in mice abolishes directed cell migration during retinal angiogenesis.\",\n      \"method\": \"BRET-based interaction assay, cell-surface expression assay, Rab4-vesicle trafficking analysis, RAMP3 knockout mouse retinal angiogenesis model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (BRET, trafficking, KO mouse phenotype), mechanistic detail on Rab4 recycling pathway\",\n      \"pmids\": [\"31712427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RAMP3 knockout mice develop normally without major vascular abnormalities, but display delayed drainage of subcutaneous lymphatic vessels (shown by ICG lymphangiography), delayed intestinal chyle transport, more severe interstitial edema in a tail lymphedema model, and impaired migration of isolated lymphatic endothelial cells in scratch-wound assays; AM administration failed to rescue re-endothelialization in RAMP3-/- cells.\",\n      \"method\": \"RAMP3 knockout mouse generation, ICG lymphangiography, tail lymphedema model, scratch-wound assay with isolated lymphatic endothelial cells\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with multiple in vivo and in vitro functional readouts, mechanistic link to AM-RAMP3 signaling in lymphatic biology\",\n      \"pmids\": [\"25264174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In a pressure-overload (TAC) model, RAMP3 knockout mice show reduced systolic function and enhanced fibrosis appearing after 4 weeks (later than RAMP2-deficient mice), with a characteristic reduction in cardiac lymphatic vessels, indicating the AM-RAMP3 system is required for later cardiac adaptation to stress through regulation of lymphatic vessels.\",\n      \"method\": \"Cardiomyocyte-specific and global RAMP3 knockout mice subjected to transverse aortic constriction, echocardiography, histological fibrosis assessment, cardiac lymphatic vessel analysis\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with multiple in vivo cardiac functional readouts, mechanistic differentiation from RAMP2 pathway in same study\",\n      \"pmids\": [\"33545715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RAMP3 deficiency in mice suppresses tumor metastasis by reducing podoplanin (PDPN)-positive cancer-associated fibroblasts (CAFs) at metastatic sites; RAMP3 was shown to mediate Src/Cas/PDPN signaling, and RAMP3-deficient CAFs displayed suppressed migration, proliferation, and metastasis in co-culture with tumor cells.\",\n      \"method\": \"RAMP3 knockout mice with splenic injection pancreatic cancer model, immunostaining for PDPN+ CAFs, in vitro co-culture assays, signaling pathway analysis (Src/Cas/PDPN)\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse model with in vivo metastasis readout and mechanistic in vitro follow-up, single lab\",\n      \"pmids\": [\"31754214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PTH induces RAMP3 mRNA expression as a primary response gene in primary mouse osteoblasts primarily through the cAMP-PKA signaling pathway; this was demonstrated by showing that cycloheximide does not block PTH-induced RAMP3 expression (primary response), forskolin mimics the effect, and the PKA inhibitor H89 blocks both PTH- and forskolin-induced RAMP3 upregulation.\",\n      \"method\": \"RT-PCR in primary mouse osteoblasts, pharmacological dissection with PKA inhibitor (H89), PKC activator (PMA), calcium ionophore (ionomycin), and protein synthesis inhibitor (cycloheximide)\",\n      \"journal\": \"Calcified tissue international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pharmacological tools dissecting signaling pathway, primary cell system, single lab\",\n      \"pmids\": [\"16075364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RAMP3 associates with CALCRL (CRLR) in vascular smooth muscle cells to mediate ADM-induced cAMP production; knockdown of RAMP3 (but not RAMP2) inhibited ADM-induced cAMP production, and proximity ligation assay showed ADM increases CALCRL-RAMP3 association; RAMP3 knockdown inhibited ADM interaction with CALCRL.\",\n      \"method\": \"siRNA knockdown of RAMP2 or RAMP3 in mesenteric artery VSMCs, cAMP assay, proximity ligation assay\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown plus proximity ligation assay for direct protein interaction, functional readout, single lab\",\n      \"pmids\": [\"26423127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In AMY3 receptors (CTR/RAMP3 heterodimers), RAMP3 fundamentally alters the dynamic role of CTR extracellular loops 2 and 3 in signal propagation compared to CTR alone; alanine scanning mutagenesis of ECL2 and ECL3 showed distinct peptide-specific effects on cAMP accumulation and ERK phosphorylation, demonstrating RAMP3 changes the signaling landscape of CTR in a peptide-dependent manner.\",\n      \"method\": \"Alanine scanning mutagenesis of CTR ECL2 and ECL3 in the context of RAMP3 co-expression, cAMP accumulation assay, ERK phosphorylation assay\",\n      \"journal\": \"ACS pharmacology & translational science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic mutagenesis with two orthogonal signaling readouts, single lab\",\n      \"pmids\": [\"32219220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RAMP3 is an early transcriptional target gene of the canonical Wnt pathway; stimulation of C57MG cells with Wnt-3a induced RAMP3 mRNA expression between 1 and 6 hours post-stimulation.\",\n      \"method\": \"Wnt-3a stimulation of C57MG cells, temporal mRNA expression analysis\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method (RT-PCR), no mechanistic follow-up on RAMP3 protein function\",\n      \"pmids\": [\"19353769\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RAMP3 knockout mice on high-fat diet show glucose intolerance and altered amylin anorectic responses; RAMP3 KO mice respond to salmon calcitonin (CTR agonist) but not amylin for food intake suppression, establishing RAMP3 as necessary for amylin's anorectic effect and glucose homeostasis mediated through the CTR/RAMP3 (AMY3) receptor.\",\n      \"method\": \"RAMP3 global KO mice, high-fat diet feeding, glucose tolerance tests, food intake pharmacology with amylin and salmon calcitonin, c-Fos immunostaining in area postrema\",\n      \"journal\": \"Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with multiple metabolic and pharmacological readouts, single lab\",\n      \"pmids\": [\"31881259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RAMP3 deficiency in ovariectomized (postmenopausal) mice exacerbates obesity, adipose tissue weight gain, insulin resistance, and hepatic steatosis; GPR30 (membrane estrogen receptor) expression was downregulated in periuterine WAT from RAMP3-/- OVX mice, suggesting a GPR30-RAMP3 interaction in metabolic regulation.\",\n      \"method\": \"RAMP3 KO mice subjected to ovariectomy plus high-fat diet, metabolic phenotyping, adipose gene expression analysis\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — KO phenotype with expression data suggesting GPR30-RAMP3 link, no direct biochemical demonstration of interaction\",\n      \"pmids\": [\"30385288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RAMP3 binds to atypical chemokine receptor 2 (ACKR2) in HCC tumor cells and promotes membrane distribution of ACKR2 through RAB4-positive vesicles, thereby facilitating CCL2 scavenging by ACKR2 and inhibiting STAT3 and AKT phosphorylation; RAMP3 also suppressed CD11b+/Gr-1+ myeloid cell infiltration and neovascularization in tumors through ACKR2.\",\n      \"method\": \"Co-IP/protein-protein interaction assays in HCC cell lines (Huh7, HepG2, Hepa1-6), RAB4 vesicle trafficking assay, syngeneic mouse tumor model, CCL2 measurement, STAT3/AKT phosphorylation assay\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding assay combined with in vivo syngeneic tumor model and signaling readouts, mechanistically consistent with RAMP3-ACKR3 interaction previously described\",\n      \"pmids\": [\"39437486\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RAMP3 interacts with GLP-1R and biases its signaling toward Ca2+ mobilization away from canonical cAMP-driven signaling; RAMP3 reduces Gαs activation but increases secondary coupling to Gαq and Gαi, leading to enhanced glucose-stimulated insulin secretion in cells overexpressing RAMP3; RAMP3 KO mice show reduced sensitivity to GLP-1 in glucose and insulin tolerance tests.\",\n      \"method\": \"BRET interaction assay, G protein coupling assays, Ca2+ mobilization assay, cAMP assay, glucose-stimulated insulin secretion in cells, RAMP3 KO mouse glucose/insulin tolerance tests\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal signaling assays plus KO mouse phenotype, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.01.24.634724\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The AMY3R (CTR/RAMP3) has a stronger transmembrane domain interface than AMY1R/AMY2R, yielding a more stable heterodimer; human and salmon calcitonin agonists promote AMY3R dissociation whereas amylin promotes AMY1/2R subunit association; these distinct subunit equilibriums modulated by agonists determine cAMP signaling output.\",\n      \"method\": \"Novel biochemical assay resolving AMYR heterodimers and free subunits, live cell membrane assays, G protein coupling assays, cAMP signaling assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — novel biochemical reconstitution-type assay with multiple signaling readouts, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.10.09.617487\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"RAMP3 is a single-transmembrane accessory protein that functions as a molecular chaperone and allosteric modulator of multiple GPCRs: it heterodimerizes with CLR (CRLR) to form the AM2 adrenomedullin receptor (with N-glycosylation and six cysteine disulfide bonds in its extracellular domain critical for function), associates with CTR to form the AMY3 amylin receptor (with RAMP3 altering ECL2/ECL3 signal propagation and stabilizing the heterodimer), interacts with ACKR3 and ACKR2 to promote Rab4-dependent rapid receptor recycling to the plasma membrane (enabling chemotactic gradient formation), and interacts with GLP-1R to bias signaling toward Gαq/Gαi and Ca2+ mobilization enhancing insulin secretion; in vivo, RAMP3 is specifically required for lymphatic vessel drainage, later cardiac adaptation to pressure overload, directed retinal angiogenesis, and the anorectic/glucose homeostatic effects of amylin.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RAMP3 is a single-transmembrane accessory protein that determines the pharmacology, trafficking, and signaling output of multiple G protein-coupled receptors by heterodimerizing with them at the cell surface [#0, #2, #6]. Its extracellular N-terminal domain forms the ligand interface of the CLR/RAMP3 (AM2) adrenomedullin receptor: residues 59-65 and Glu74 are critical for high-affinity adrenomedullin binding and AM-evoked cAMP production, and N-glycosylation together with six cysteine-stabilized disulfide bonds are required for functional receptor expression [#0, #1, #4]. RAMP3 identity dictates the antagonist selectivity of the receptor complex, distinguishing CLR/RAMP3 from CLR/RAMP2 pharmacologically [#2], and helix 1 and helix 2 residues govern CLR association and trafficking versus ligand-pocket formation [#3]. Beyond CLR, RAMP3 partners with the calcitonin receptor (CTR) to form the AMY3 amylin receptor, where it reshapes the contribution of CTR extracellular loops 2 and 3 to signal propagation and stabilizes a transmembrane-domain heterodimer interface whose agonist-driven dissociation tunes cAMP output [#12, #18]. RAMP3 also acts as a recycling chaperone for the atypical chemokine receptors ACKR3 and ACKR2, promoting their Rab4-dependent return to the plasma membrane to sustain chemokine scavenging and chemotactic gradient formation [#6, #16], and biases GLP-1R signaling away from Gαs/cAMP toward Gαq/Gαi and Ca2+ mobilization to enhance glucose-stimulated insulin secretion [#17]. In vivo, RAMP3 is dispensable for baseline vascular development but is specifically required for lymphatic vessel drainage, directed retinal angiogenesis, later cardiac adaptation to pressure overload through cardiac lymphatics, and the anorectic and glucose-homeostatic effects of amylin [#6, #7, #8, #14].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established which part of RAMP3 contacts ligand, answering how an accessory protein contributes to adrenomedullin pharmacology rather than merely escorting the receptor.\",\n      \"evidence\": \"Deletion mutagenesis with 125I-AM binding and cAMP readouts in HEK293 cells co-expressing CRLR\",\n      \"pmids\": [\"11591721\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not resolve the structural fold of the bound complex\", \"No information on RAMP3 partners beyond CLR\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the structural requirements (N-glycosylation, disulfide bonds) for a functional CLR/RAMP3 receptor and showed RAMP identity dictates antagonist pharmacology.\",\n      \"evidence\": \"Oocyte and COS7 reconstitution with cysteine/glycosylation mutagenesis and cross-species pharmacological antagonism\",\n      \"pmids\": [\"12939163\", \"12970090\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Disulfide connectivity not directly mapped\", \"Species-dependence of antagonist selectivity not mechanistically explained\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed RAMP3 expression is regulated, identifying it as a cAMP-PKA-driven primary response gene downstream of PTH in osteoblasts.\",\n      \"evidence\": \"RT-PCR with cycloheximide, forskolin, and H89 pharmacological dissection in primary mouse osteoblasts\",\n      \"pmids\": [\"16075364\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcription factors mediating induction not identified\", \"Functional consequence of RAMP3 induction in bone not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Separated the RAMP determinants of receptor association/trafficking from those shaping the ligand-binding pocket, refining how RAMP3-type proteins remodel CLR.\",\n      \"evidence\": \"RAMP1/RAMP3 chimera analysis with CLR trafficking and agonist potency assays plus molecular modeling\",\n      \"pmids\": [\"20017504\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Modeling-based, no experimental structure\", \"Single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Pinpointed a single residue (Glu74) contributing charge/geometry to adrenomedullin specificity at the AM2 receptor.\",\n      \"evidence\": \"Site-directed mutagenesis of RAMP3 with cAMP and surface-ELISA readouts in COS7 cells\",\n      \"pmids\": [\"21402116\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single residue context only\", \"No structural confirmation of the contact\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Moved RAMP3 from in vitro pharmacology to physiology, showing it is selectively required for lymphatic vessel function rather than general vascular development.\",\n      \"evidence\": \"RAMP3 knockout mice with ICG lymphangiography, tail lymphedema model, and lymphatic endothelial scratch-wound assays\",\n      \"pmids\": [\"25264174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor partner mediating the lymphatic phenotype not isolated in vivo\", \"Cell-autonomous versus systemic contributions not separated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed a chaperone/recycling function for RAMP3 distinct from G-protein modulation, explaining how it controls chemokine receptor surface levels and gradient formation.\",\n      \"evidence\": \"BRET interaction, Rab4-vesicle trafficking analysis, and RAMP3 KO mouse retinal angiogenesis\",\n      \"pmids\": [\"31712427\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Recruitment mechanism of RAMP3 to Rab4 vesicles unknown\", \"Generality of the recycling role across other GPCRs untested at the time\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended RAMP3 function to AMY3 (CTR/RAMP3) signaling and to tumor-stromal biology, showing peptide-specific reshaping of CTR extracellular-loop signaling and a metastasis-promoting role via CAFs.\",\n      \"evidence\": \"Alanine scanning of CTR ECL2/ECL3 with cAMP and ERK readouts; RAMP3 KO mouse metastasis model with Src/Cas/PDPN signaling analysis\",\n      \"pmids\": [\"32219220\", \"31754214\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct structural basis of ECL remodeling not resolved\", \"Mechanistic link between RAMP3 GPCR activity and Src/Cas/PDPN in CAFs not fully traced\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked RAMP3 to organ-level stress adaptation, showing its loss impairs late cardiac compensation to pressure overload via reduced cardiac lymphatics.\",\n      \"evidence\": \"Global and cardiomyocyte-specific RAMP3 KO mice with transverse aortic constriction, echocardiography, fibrosis histology, and lymphatic vessel analysis\",\n      \"pmids\": [\"33545715\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Temporal divergence from RAMP2 pathway mechanistically unexplained\", \"Direct ligand-receptor axis in cardiac lymphatics not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established RAMP3 as physiologically required for amylin's anorectic and glucose-homeostatic actions through the AMY3 receptor.\",\n      \"evidence\": \"RAMP3 global KO mice on high-fat diet with glucose tolerance tests, amylin/salmon calcitonin food-intake pharmacology, and area postrema c-Fos staining\",\n      \"pmids\": [\"31881259\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Central versus peripheral site of action not fully resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Generalized the Rab4-recycling chaperone mechanism to ACKR2, connecting RAMP3 to CCL2 scavenging and an anti-tumor immune/vascular phenotype.\",\n      \"evidence\": \"Co-IP, RAB4 trafficking, syngeneic HCC tumor model, CCL2 measurement, and STAT3/AKT phosphorylation assays\",\n      \"pmids\": [\"39437486\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding interface with ACKR2 not mapped\", \"Reconciliation with metastasis-promoting role in other models unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed RAMP3 acts as a signaling-bias modulator of GLP-1R, redirecting it from Gαs/cAMP toward Gαq/Gαi and Ca2+ to enhance insulin secretion.\",\n      \"evidence\": \"BRET interaction, G protein coupling and Ca2+/cAMP assays, glucose-stimulated insulin secretion, and RAMP3 KO mouse tolerance tests (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.01.24.634724\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Structural basis of biased coupling not determined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided a biophysical basis for AMY3 receptor behavior, showing a stronger TM interface and agonist-dependent subunit equilibrium that tunes signaling.\",\n      \"evidence\": \"Novel heterodimer-resolving biochemical assay with live-cell membrane and G protein/cAMP readouts (preprint)\",\n      \"pmids\": [\"bio_10.1101_2024.10.09.617487\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"In vivo relevance of the dissociation equilibrium untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RAMP3 selects among its many GPCR partners in a given cell, and whether its recycling-chaperone versus pharmacology-modulating roles are coordinated, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of RAMP3 in complex with non-CLR partners reported in the corpus\", \"Determinants of partner choice in native tissues unknown\", \"Mechanism coupling RAMP3 expression regulation to its receptor functions undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2, 12, 17]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [6, 16, 17]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 6, 16]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [6, 16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 6, 12, 17]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [6, 16]}\n    ],\n    \"complexes\": [\"CLR/RAMP3 (AM2) receptor\", \"CTR/RAMP3 (AMY3) amylin receptor\"],\n    \"partners\": [\"CALCRL\", \"CALCR\", \"ACKR3\", \"ACKR2\", \"GLP1R\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}