{"gene":"RAMP3","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":1998,"finding":"RAMP3, together with RAMP1 and RAMP2, forms a family of single-transmembrane-domain proteins required to transport the calcitonin receptor-like receptor (CRLR) to the plasma membrane; RAMP2- or RAMP3-transported CRLR functions as an adrenomedullin receptor, whereas RAMP1-transported CRLR functions as a CGRP receptor.","method":"Transfection/co-expression in cell lines with radioligand binding and functional cAMP assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — foundational discovery paper replicated widely across labs, multiple orthogonal methods","pmids":["9620797"],"is_preprint":false},{"year":1999,"finding":"RAMP3 co-expressed with the calcitonin receptor (CTR) generates a distinct amylin receptor phenotype (AMY3R) with reduced efficacy for human CGRPα compared to the RAMP1/CTR (AMY1R) combination; RAMP3 is located intracellularly when expressed alone but traffics to the cell surface upon co-expression with CTR.","method":"Co-transfection in COS-7 cells, 125I-amylin radioligand binding competition, cAMP functional assays, confocal microscopy with c-myc-tagged RAMP1, and cross-linking experiments","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods in a single rigorous study","pmids":["10385705"],"is_preprint":false},{"year":2000,"finding":"CRLR is endocytosed together with RAMP3 (and RAMP1/2) via clathrin-coated vesicles following agonist stimulation; internalized CRLR/RAMP complexes are largely targeted to a degradative (lysosomal) pathway with inefficient recycling, and CRLR cannot reach the plasma membrane without RAMPs.","method":"Stable expression of CRLR-GFP chimera ± RAMPs in HEK293 cells; agonist-stimulated internalization tracked by fluorescence microscopy; hypertonic sucrose inhibition of clathrin-dependent endocytosis; co-localization with rhodamine-transferrin and lysosomal markers","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — live-cell imaging with functional validation and pharmacological controls","pmids":["10882736"],"is_preprint":false},{"year":2001,"finding":"Direct protein–protein interaction between RAMP3 (or RAMP2) and CRLR, not differential glycosylation, determines ligand-binding selectivity: cross-linking experiments showed that 125I-AM is incorporated into RAMP2 and RAMP3 within the CRLR-RAMP heterodimer, placing the RAMPs close to the peptide-binding pocket; fully glycosylated CRLR, not the core-glycosylated form produced by RAMP2/3 co-expression, represents the functional AM-binding receptor.","method":"Co-expression in HEK293 cells, cross-linking with bis-succinimidyl suberate, radioligand binding (125I-AM, 125I-CGRP), glycosylation analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — biochemical reconstitution with cross-linking and radioligand binding, multiple orthogonal methods","pmids":["11387328"],"is_preprint":false},{"year":2001,"finding":"Seven specific amino acids in the extracellular N-terminal domain of RAMP3 (residues 59–65) are critical for high-affinity AM binding and AM-evoked cAMP production at the CRLR/RAMP3 adrenomedullin receptor; deletion of this segment significantly attenuates 125I-AM binding despite preserved cell-surface expression of the receptor heterodimer.","method":"RAMP chimeras and deletion mutants co-expressed with CRLR in HEK293 cells; 125I-AM radioligand binding; AM-evoked cAMP production assay; cell-surface expression confirmed by ELISA","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis combined with radioligand binding and functional cAMP assay","pmids":["11591721"],"is_preprint":false},{"year":2002,"finding":"RAMP3 interacts with the PTH2 receptor (in addition to CRLR and CTR), extending the known repertoire of RAMP3 receptor partners to at least one additional class II GPCR; RAMP-receptor complexes beyond CL/CTR can modulate downstream signaling.","method":"Immunofluorescence confocal microscopy co-localization; radioligand binding in COS-7 cells; phosphoinositide hydrolysis assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 — single-lab co-localization study with partial functional follow-up","pmids":["12446722"],"is_preprint":false},{"year":2003,"finding":"N-glycosylation of RAMP3 (at two to four of its four consensus sites) and disulfide bonds formed by all six conserved cysteine residues in its extracellular domain are each essential for functional expression of the CRLR/RAMP3 adrenomedullin receptor: elimination of all N-glycans reduces 125I-AM binding and increases EC50, while mutation of all cysteines to serine completely abolishes AM binding.","method":"Xenopus oocyte expression system; site-directed mutagenesis of N-glycosylation sites and cysteines; 125I-AM radioligand binding; EC50 determination for AM-evoked responses","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis with radioligand binding and functional assay in a well-controlled expression system","pmids":["12939163"],"is_preprint":false},{"year":2003,"finding":"CL/RAMP2 and CL/RAMP3 receptors are pharmacologically distinct: AM22-52 selectively antagonizes CL/RAMP2 over CL/RAMP3 in human receptor combinations, whereas CGRP8-37 is an effective antagonist at CL/RAMP3 but shows species-dependent effects at CL/RAMP2; the agonist potency order at CL/RAMP3 is AM ≈ βCGRP > αCGRP.","method":"Transient transfection in COS-7 cells and endogenous receptor expression in Rat 2 and L6 cells; cAMP functional assays with peptide antagonists; apparent pA2 determination","journal":"British journal of pharmacology","confidence":"High","confidence_rationale":"Tier 2 — pharmacological characterization across multiple receptor combinations and cell systems","pmids":["12970090"],"is_preprint":false},{"year":2004,"finding":"The C-terminal PDZ type I motif unique to RAMP3 (absent from RAMP1 and RAMP2) mediates interaction with N-ethylmaleimide-sensitive factor (NSF), redirecting the CRLR/RAMP3 (AM2R) complex from a degradative pathway to a recycling pathway after agonist-stimulated internalization; deletion or point mutation of the PDZ motif abolishes this NSF interaction and recycling.","method":"Adenylate cyclase assays, radioligand binding, immunofluorescence microscopy in HEK293 cells; mutational analysis (deletion and point mutations of PDZ motif); RNA interference of RAMP3 in rat mesangial cells; pharmacological inhibition of NSF","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis + RNAi + pharmacological inhibition with multiple functional readouts, replicated in endogenous cell system","pmids":["15613468"],"is_preprint":false},{"year":2005,"finding":"Parathyroid hormone (PTH) induces RAMP3 mRNA expression in primary mouse osteoblasts as a primary response gene (cycloheximide-insensitive) primarily through the cAMP–PKA signaling pathway; PKA inhibition with H89 blocks PTH- and forskolin-induced RAMP3 upregulation, whereas PKC activation with PMA also increases RAMP3 but calcium signaling (ionomycin) does not.","method":"Primary mouse osteoblast cultures; representational difference analysis; RT-PCR for RAMP3 mRNA; pharmacological dissection using cycloheximide, forskolin, H89 (PKA inhibitor), PMA (PKC activator/depletor), ionomycin, and PTH(3-34)","journal":"Calcified tissue international","confidence":"Medium","confidence_rationale":"Tier 3 — pharmacological pathway dissection in primary cells, single lab","pmids":["16075364"],"is_preprint":false},{"year":2008,"finding":"Residue 74 in RAMP3 (Glu74) is a key determinant of AM pharmacology at the CL/RAMP3 (AM2) receptor; substitution of RAMP3 residues into RAMP1 and vice versa at position 74 converts AM potency profiles, with RAMP1 Phe93 additionally contributing to αCGRP potency at the CGRP receptor.","method":"Site-directed mutagenesis of RAMP1 and RAMP3 at eight conserved positions; co-transfection with CL or CTR in COS-7 cells; agonist-stimulated cAMP production; cell-surface expression by ELISA","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 1 — systematic mutagenesis with functional and expression readouts","pmids":["18593822"],"is_preprint":false},{"year":2009,"finding":"RAMP3 is an early Wnt target gene: in C57MG cells stimulated with Wnt-3a, RAMP3 mRNA is induced within 1–6 hours, classifying it as an early (not immediate or late) transcriptional target of canonical Wnt/β-catenin signaling.","method":"Wnt-3a stimulation of C57MG cells; time-course RT-PCR for RAMP3 and other Wnt target genes; comparison of induction kinetics","journal":"Cell biology international","confidence":"Medium","confidence_rationale":"Tier 3 — single-lab expression kinetics study; mechanistic implication (Wnt regulation of RAMP3) supported by pathway context","pmids":["19353769"],"is_preprint":false},{"year":2010,"finding":"Structure–function analysis of RAMP1–RAMP3 chimeras reveals that extracellular helix 1 and residues 62–69 of helix 2 are required for CRLR trafficking (CLR association), while residues 86–89 at the helix 2/3 junction influence peptide (αCGRP and AM) binding potency without affecting trafficking; RAMP3 chimera data suggest the helix 2/3 loop is less exposed than in RAMP1 and plays a role in peptide binding.","method":"RAMP1/RAMP3 chimera co-expression with CLR or CTR in COS-7 cells; CLR trafficking assay; agonist-stimulated cAMP production; homology modeling","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — chimeric mutagenesis with trafficking and functional readouts, modeling provides mechanistic context","pmids":["20017504"],"is_preprint":false},{"year":2011,"finding":"Glu74 in RAMP3 specifically contributes to AM potency at the AM2 (CL/RAMP3) receptor through geometry and charge: Phe substitution reduces AM potency while Tyr does not, and most other substitutions have small effects; the equivalent residue Trp74 in RAMP1 has distinct behavior (Tyr enhances AM potency), confirming position 74 as a key differential determinant between AM2 and CGRP receptors.","method":"Site-directed mutagenesis of Glu74 in RAMP3 and Trp74 in RAMP1 with multiple amino acid substitutions; co-expression with CLR in COS-7 cells; cAMP assay for AM, AM2/intermedin, and CGRP; cell-surface expression by ELISA","journal":"Peptides","confidence":"High","confidence_rationale":"Tier 1 — systematic mutagenesis with functional and expression readouts confirming prior findings","pmids":["21402116"],"is_preprint":false},{"year":2013,"finding":"RAMP3 physically interacts with GPR30 (G-protein-coupled estrogen receptor 1/GPER1): BRET titration, co-immunoprecipitation, and confocal microscopy demonstrate direct interaction; GPR30 presence increases RAMP3 plasma membrane expression in HEK293 cells; in vivo, RAMP3 knockout disrupts GPR30 subcellular localization in cardiac cells and abolishes GPR30-agonist (G-1)-mediated reduction of cardiac hypertrophy and perivascular fibrosis in a sex-dependent manner.","method":"BRET titration assays, co-immunoprecipitation, confocal microscopy in HEK293 cells; in vivo RAMP3 knockout mice on heart disease-prone background treated with G-1; cardiac histopathology","journal":"Journal of molecular endocrinology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal in vitro methods plus in vivo validation with defined phenotypic readout","pmids":["23674134"],"is_preprint":false},{"year":2014,"finding":"RAMP3 knockout mice are viable and display normal postnatal blood vessel angiogenesis, but exhibit delayed lymphatic drainage (shown by ICG lymphangiography), delayed chyle transport by intestinal lymphatics, more severe interstitial edema in a tail lymphedema model, and a primary migration defect in isolated RAMP3−/− lymphatic endothelial cells that is not rescued by AM; RAMP2 knockout (but not RAMP3 knockout) is embryonically lethal due to vascular defects, establishing distinct functional roles for the two AM receptor subtypes.","method":"RAMP2−/− and RAMP3−/− mouse generation; ICG lymphangiography; tail lymphedema model with histology; in vitro scratch-wound migration assay of isolated lymphatic endothelial cells","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with multiple orthogonal in vivo and in vitro phenotypic readouts","pmids":["25264174"],"is_preprint":false},{"year":2015,"finding":"In rat mesenteric artery smooth muscle cells (VSMCs), RAMP3 (not RAMP2) associates with CALCRL to mediate ADM-induced cAMP production; RAMP3 knockdown inhibits ADM-induced cAMP generation and ADM–CALCRL association; proximity ligation assay directly demonstrates RAMP3–CALCRL co-localization enhanced by ADM.","method":"RAMP2/RAMP3 siRNA knockdown in VSMCs; cAMP production assay with peptide antagonists; proximity ligation assay for RAMP3–CALCRL association","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 — RNAi knockdown and proximity ligation in primary-like cells, single lab","pmids":["26423127"],"is_preprint":false},{"year":2019,"finding":"RAMP3 interacts 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; RAMP3–ACKR3 interaction reduces AM ligand availability without changing G-protein coupling; genetic deletion of RAMP3 in mice abolishes directed retinal angiogenesis, phenocopying ACKR3 deletion.","method":"BRET-based and cell-surface expression screens for RAMP–chemokine receptor interactions; Rab4-positive vesicle trafficking assays; AM ligand scavenging assays; RAMP3 and ACKR3 global knockout mice; retinal angiogenesis imaging","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — comprehensive BRET screen, mechanistic trafficking assays, and in vivo genetic validation with defined phenotype","pmids":["31712427"],"is_preprint":false},{"year":2019,"finding":"RAMP3 deficiency in cancer-associated fibroblasts (CAFs) suppresses tumor metastasis by reducing podoplanin (PDPN)-positive CAF numbers at metastatic sites; RAMP3 regulates PDPN expression via Src/Cas/PDPN signaling in fibroblasts; RAMP3−/− CAFs show reduced migration, proliferation, and metastasis-promoting activity in co-cultures with tumor cells in vitro and in vivo.","method":"Splenic injection of PAN02 pancreatic cancer cells in RAMP3−/− and DI-E-RAMP2−/− mice for spontaneous liver metastasis model; podoplanin immunohistochemistry; Src/Cas pathway analysis; in vitro co-culture migration and proliferation assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo KO model with mechanistic pathway analysis (Src/Cas/PDPN) and in vitro corroboration, single lab","pmids":["31754214"],"is_preprint":false},{"year":2019,"finding":"RAMP3 knockout mice display glucose intolerance (on chow diet in males) and altered meal patterns (decreased intermeal interval); amylin and salmon calcitonin anorectic effects are mediated through distinct RAMP-containing receptor combinations: RAMP1/3 double KO mice are insensitive to both amylin and sCT, while RAMP3 KO mice respond to sCT but not amylin, indicating RAMP3 specifically mediates part of amylin's anorectic signaling.","method":"Global RAMP1, RAMP3, and RAMP1/3 double KO mice; glucose tolerance tests; intraperitoneal amylin and sCT injection with food intake measurement; meal pattern analysis; c-Fos immunohistochemistry in area postrema","journal":"Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO with multiple metabolic readouts, single lab","pmids":["31881259"],"is_preprint":false},{"year":2019,"finding":"RAMP3 alanine scanning of extracellular loops (ECL2 and ECL3) of the calcitonin receptor in the AMY3R context demonstrates that RAMP3 co-expression fundamentally alters the dynamic role of ECL2 and ECL3 in propagating cAMP and ERK signaling compared to CTR alone; the effects are highly peptide-dependent (sCT, hCT, pCT, rat amylin, hCGRP show distinct ECL mutation effects in the AMY3R context).","method":"Alanine scanning mutagenesis of CTR ECL2 and ECL3 co-expressed with RAMP3; cAMP accumulation assay; ERK phosphorylation (pERK) assay; cell-surface expression by ELISA","journal":"ACS pharmacology & translational science","confidence":"Medium","confidence_rationale":"Tier 1 — systematic mutagenesis with two signaling readouts, single lab","pmids":["32219220"],"is_preprint":false},{"year":2020,"finding":"Cryo-EM structures of the AM2R (CL/RAMP3) in complex with AM or intermedin and Gs protein reveal that RAMP3 positions its extracellular domain (ECD) at a distinct primary orientation relative to the receptor core compared to RAMP2 in AM1R, and that the RAMP linker region connecting the TM helix and ECD controls receptor phenotype; distinct ECL3 positioning and ECD mobility are receptor-dependent features.","method":"Cryo-EM structure determination of AM2R–AM–Gs (2.4 Å) and AM2R–AM2–Gs (2.3 Å); chimeric RAMP linker exchange experiments; dynamic analysis of cryo-EM micrographs","journal":"ACS pharmacology & translational science","confidence":"High","confidence_rationale":"Tier 1 — high-resolution cryo-EM structures with chimeric functional validation","pmids":["32296767"],"is_preprint":false},{"year":2021,"finding":"Cardiomyocyte-specific RAMP3 knockout mice show normal baseline cardiac function but develop reduced systolic function and enhanced fibrosis after 4 weeks of transverse aortic constriction (TAC), with a characteristic reduction in cardiac lymphatic vessels, indicating that the AM–RAMP3 system is required for later adaptive cardiac responses to cardiovascular stress through regulation of lymphatic vessels.","method":"Cardiomyocyte-specific RAMP3 KO mice; TAC model; echocardiography; histological fibrosis quantification; cardiac lymphatic vessel immunostaining","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — tissue-specific KO with defined phenotypic readout, single lab","pmids":["33545715"],"is_preprint":false},{"year":2022,"finding":"Cryo-EM structures of AMY3R (CTR/RAMP3) with amylin and other calcitonin family peptides demonstrate that RAMP3 constrains the conformation of amylin-bound AMY3R through a midpeptide 'bypass motif', and that CT-based peptide activation of AMY3R is structurally distinct from amylin-based activation; these structural differences have direct implications for ligand selectivity.","method":"Cryo-EM structure determination of AMY3R–amylin, AMY1R–sCT, AMY2R–sCT/hCT, and CTR–amylin/sCT/hCT complexes; structural comparison and dynamics analysis","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — high-resolution cryo-EM structures of multiple complexes with rigorous comparative analysis","pmids":["35324283"],"is_preprint":false},{"year":2024,"finding":"RAMP3 binds atypical chemokine receptor 2 (ACKR2) in HCC tumor cells and promotes ACKR2 membrane distribution through Rab4-positive vesicles, enabling ACKR2-mediated CCL2 scavenging/degradation; RAMP3 expression reduces intratumoral CCL2, inhibits STAT3 and AKT phosphorylation, and reduces CD11b+/Gr-1+ myeloid cell infiltration and neovascularization in syngeneic tumors through ACKR2.","method":"Co-IP and bioinformatic PPI analysis; overexpression and knockdown in Huh7, HepG2, and Hepa1-6 cells; syngeneic tumor implantation; CCL2 ELISA; STAT3/AKT phosphorylation Western blot; flow cytometry for myeloid infiltration; immunohistochemistry for neovascularization","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple in vitro and in vivo methods, single lab, extends ACKR interaction repertoire of RAMP3","pmids":["39437486"],"is_preprint":false},{"year":2025,"finding":"RAMP3 interacts with the GLP-1 receptor (GLP-1R) and biases its signaling: RAMP3 co-expression reduces canonical Gαs/cAMP coupling while increasing Gαq and Gαi coupling, shifting the receptor toward Ca2+ mobilization; this altered signaling profile elevates glucose-stimulated insulin secretion in cells overexpressing RAMP3, and RAMP3 knockout mice show reduced sensitivity to GLP-1 in glucose and insulin tolerance tests.","method":"BRET assays for GLP-1R–RAMP3 interaction and G-protein coupling; Ca2+ mobilization assay; cAMP assay; glucose-stimulated insulin secretion assay; RAMP3 KO mouse glucose and insulin tolerance tests","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal signaling assays plus in vivo KO validation, but preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.01.24.634724"],"is_preprint":true},{"year":2024,"finding":"AMY3R (CTR/RAMP3) has a stronger transmembrane domain interface than AMY1R or AMY2R, resulting in a more stable basal CTR–RAMP3 subunit association; calcitonin agonists (human and salmon CT) promote AMY3R dissociation, while rat amylin promotes AMY1/2R subunit association; these altered equilibria directly affect G-protein coupling and cAMP signaling output.","method":"Novel biochemical assay resolving AMYR heterodimers vs. free subunits; live-cell membrane subunit association assays; G-protein coupling assays; cAMP signaling assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — novel biochemical approach with multiple functional readouts, but 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 heterodimerizes with multiple GPCRs (primarily CLR and CTR, but also ACKR3, ACKR2, GPR30, PTH2R, and GLP-1R) to act as a molecular chaperone promoting cell-surface expression, a determinant of ligand-binding pharmacology through its extracellular N-terminal domain (particularly residues 59–65 and Glu74), and a regulator of post-endocytic receptor trafficking via its unique C-terminal PDZ motif that recruits NSF to redirect internalized receptors toward recycling rather than degradation; in vivo, RAMP3 is required for lymphatic vessel function, guided angiogenesis through ACKR3-dependent chemotactic gradient formation, sex-dependent GPR30-mediated cardioprotection, glucose homeostasis, and amylin-mediated satiation signaling."},"narrative":{"teleology":[{"year":1998,"claim":"Identification of RAMP3 as part of a new family of single-TM accessory proteins required for CRLR plasma membrane delivery and ligand-specificity switching (CGRP vs. AM receptor) resolved the longstanding paradox of how a single receptor generated two pharmacologically distinct binding phenotypes.","evidence":"Co-expression in cell lines with radioligand binding and cAMP assays","pmids":["9620797"],"confidence":"High","gaps":["Stoichiometry and direct physical interaction not yet demonstrated","Mechanism of selectivity switch unknown"]},{"year":1999,"claim":"Demonstration that RAMP3 also partners with the calcitonin receptor to generate a distinct amylin receptor (AMY3R) established that RAMPs are not CRLR-specific accessories but general GPCR modulators, and showed RAMP3 requires a receptor partner for surface expression.","evidence":"Co-transfection in COS-7 cells with radioligand binding, cAMP assays, and confocal microscopy","pmids":["10385705"],"confidence":"High","gaps":["Full repertoire of RAMP3-interacting GPCRs unknown","Structural basis of AMY3R vs. AMY1R selectivity unresolved"]},{"year":2001,"claim":"Cross-linking and mutagenesis studies established that RAMP3 directly contacts the ligand-binding pocket and that residues 59–65 in its extracellular domain are critical for AM binding, shifting the mechanistic model from glycosylation-dependent to direct protein–protein interaction-dependent pharmacology.","evidence":"Cross-linking with 125I-AM, RAMP chimera/deletion mutagenesis with radioligand binding and cAMP assays in HEK293 cells","pmids":["11387328","11591721"],"confidence":"High","gaps":["High-resolution structure of the RAMP3–CLR interface not available","Contribution of individual residues within 59–65 not dissected"]},{"year":2003,"claim":"Post-translational requirements for RAMP3 function were defined: N-glycosylation and disulfide bonds formed by all six conserved cysteines are essential for functional AM receptor expression, and pharmacological profiling established that AM2R (CLR/RAMP3) has distinct antagonist sensitivity from AM1R (CLR/RAMP2).","evidence":"Mutagenesis in Xenopus oocytes and pharmacological characterization in COS-7 cells with cAMP assays","pmids":["12939163","12970090"],"confidence":"High","gaps":["Structure of RAMP3 ECD not solved","Species-dependent pharmacological differences not fully explained mechanistically"]},{"year":2004,"claim":"Discovery that the C-terminal PDZ motif unique to RAMP3 recruits NSF to reroute internalized CLR/RAMP3 from degradation to recycling answered why AM2R resensitizes more efficiently than AM1R and introduced a trafficking-based mechanism for differential RAMP function.","evidence":"Mutagenesis, RNAi, and pharmacological NSF inhibition in HEK293 and rat mesangial cells with cAMP, binding, and immunofluorescence readouts","pmids":["15613468"],"confidence":"High","gaps":["Identity of PDZ domain-containing scaffold linking RAMP3 to NSF not determined","Whether recycling mechanism applies to non-CLR partners unknown at this point"]},{"year":2008,"claim":"Systematic mutagenesis pinpointed Glu74 as the single residue most critical for differential AM potency between AM2R and CGRP receptor, providing the first residue-level explanation for RAMP-dependent pharmacological switching.","evidence":"Site-directed mutagenesis of eight conserved positions in RAMP1/RAMP3 with cAMP and surface expression assays","pmids":["18593822","21402116"],"confidence":"High","gaps":["Structural context of Glu74 within the peptide-binding interface not visualized","Role in intermedin/AM2 binding not fully assessed"]},{"year":2013,"claim":"RAMP3 was shown to physically interact with GPR30 (GPER1), a non-class-B GPCR, expanding the interactome well beyond CLR/CTR; in vivo, RAMP3 deletion abolished GPR30-agonist-mediated cardioprotection in a sex-dependent manner.","evidence":"BRET, co-IP, confocal in HEK293; RAMP3 KO mice with TAC and G-1 treatment; cardiac histopathology","pmids":["23674134"],"confidence":"High","gaps":["Mechanism by which RAMP3 modulates GPR30 signaling (trafficking vs. coupling) not dissected","Sex-dependence mechanism unclear"]},{"year":2014,"claim":"RAMP3 knockout mice revealed a non-redundant role for RAMP3 in lymphatic vessel function — lymphatic drainage, chyle transport, and lymphatic endothelial cell migration were all impaired — distinguishing RAMP3 from RAMP2 whose loss causes embryonic-lethal vascular defects.","evidence":"Global RAMP3 KO mice; ICG lymphangiography; tail lymphedema model; isolated lymphatic EC migration assay","pmids":["25264174"],"confidence":"High","gaps":["Which RAMP3 receptor partner mediates lymphatic effects not identified","Whether PDZ-dependent recycling is involved in lymphatic function untested"]},{"year":2019,"claim":"Three independent 2019 studies collectively broadened RAMP3 biology: (1) RAMP3 interaction with ACKR3 enables Rab4-dependent receptor recycling and AM scavenging required for retinal angiogenesis; (2) RAMP3 in cancer-associated fibroblasts regulates podoplanin via Src/Cas signaling to promote metastasis; (3) RAMP3 KO mice are glucose intolerant and lose amylin-mediated anorexia, establishing RAMP3 in metabolic homeostasis.","evidence":"BRET screens, Rab4 trafficking assays, retinal angiogenesis in KO mice; syngeneic tumor metastasis models with Src/Cas pathway analysis; metabolic phenotyping of global RAMP1/RAMP3 KO mice with amylin/sCT injection","pmids":["31712427","31754214","31881259"],"confidence":"High","gaps":["Whether RAMP3 PDZ motif mediates ACKR3 recycling specifically through NSF not shown","CAF findings from single cancer model","Brain region-specific RAMP3 function in satiation not mapped"]},{"year":2020,"claim":"Cryo-EM structures of AM2R (CLR/RAMP3) at 2.3–2.4 Å resolution revealed how RAMP3 ECD orientation and the linker region between TM and ECD control receptor phenotype, providing the first atomic-level explanation for RAMP-dependent pharmacology.","evidence":"Cryo-EM of AM2R–AM–Gs and AM2R–intermedin–Gs; chimeric RAMP linker swaps","pmids":["32296767"],"confidence":"High","gaps":["No structure of RAMP3 in complex with CTR (AMY3R) at this time","Intracellular PDZ domain region not resolved"]},{"year":2022,"claim":"Cryo-EM of AMY3R (CTR/RAMP3) showed that RAMP3 constrains amylin binding through a midpeptide bypass motif and that calcitonin-based versus amylin-based activation proceeds through structurally distinct mechanisms, explaining peptide-dependent signaling differences.","evidence":"Cryo-EM structures of AMY3R–amylin and related CTR/RAMP complexes with multiple peptides","pmids":["35324283"],"confidence":"High","gaps":["Structures are Gs-coupled only; Gq or Gi-coupled states unknown","RAMP3 C-terminal PDZ motif still not resolved structurally"]},{"year":2024,"claim":"RAMP3 was shown to promote ACKR2 membrane expression via Rab4 vesicles and enable CCL2 scavenging in hepatocellular carcinoma cells, extending the RAMP3-dependent recycling mechanism to a second atypical chemokine receptor and linking it to tumor immune microenvironment regulation.","evidence":"Co-IP, overexpression/knockdown in HCC cell lines, syngeneic tumors, CCL2 ELISA, flow cytometry for myeloid infiltration","pmids":["39437486"],"confidence":"Medium","gaps":["Whether RAMP3 PDZ motif or another domain mediates ACKR2 interaction not determined","Single tumor model","Independent replication needed"]},{"year":null,"claim":"Key unresolved questions include the structural basis of the RAMP3 C-terminal PDZ motif–NSF interaction, the full inventory of RAMP3-interacting GPCRs, and how RAMP3 biases G-protein coupling selectivity (e.g., at GLP-1R) at a structural level.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal or cryo-EM structure of the RAMP3 PDZ motif with NSF or other scaffold","Comprehensive unbiased interactome screen for RAMP3 across all GPCRs not performed","Mechanism of G-protein coupling bias by RAMP3 structurally undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,8,17,24]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,10,13,20,23]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[3,7]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,2,14,17]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2,8,17]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[8,17,24]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,7,8,10,14,20,23]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,6,8]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,8,17,24]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[15,17]}],"complexes":["AM2R (CLR/RAMP3)","AMY3R (CTR/RAMP3)","ACKR3/RAMP3","GPR30/RAMP3"],"partners":["CALCRL","CALCR","ACKR3","ACKR2","GPER1","NSF","PTH2R"],"other_free_text":[]},"mechanistic_narrative":"RAMP3 is a single-transmembrane accessory protein that heterodimerizes with multiple GPCRs to regulate their trafficking, pharmacology, and signaling. It was originally identified as a chaperone that escorts the calcitonin receptor-like receptor (CLR) to the cell surface, where CLR/RAMP3 forms the AM2 adrenomedullin receptor, with residues 59–65 and Glu74 in the RAMP3 extracellular domain determining high-affinity AM binding [PMID:9620797, PMID:11591721, PMID:18593822]. A unique C-terminal PDZ motif distinguishes RAMP3 from RAMP1/RAMP2 by recruiting NSF to redirect internalized receptor complexes from lysosomal degradation to Rab4-positive recycling endosomes, a mechanism operative for both CLR and the atypical chemokine receptors ACKR3 and ACKR2 [PMID:15613468, PMID:31712427, PMID:39437486]. In vivo, RAMP3 is required for lymphatic vessel function, ACKR3-dependent retinal angiogenesis, amylin-mediated satiation signaling, GPR30-dependent cardioprotection, and glucose homeostasis [PMID:25264174, PMID:31712427, PMID:31881259, PMID:23674134]."},"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 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cardioprotection.","date":"2013","source":"Journal of molecular endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/23674134","citation_count":61,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16237,"output_tokens":4494,"usd":0.058061},"stage2":{"model":"claude-opus-4-6","input_tokens":7959,"output_tokens":2822,"usd":0.165518},"total_usd":0.544614,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":29428,"output_tokens":7557,"usd":0.10082},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":11577,"output_tokens":3557,"usd":0.220215}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"Specific 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 attenuates 125I-AM binding and cAMP production without affecting cell surface expression of the receptor heterodimer.\",\n      \"method\": \"Chimeric RAMP constructs and deletion mutants co-expressed with CRLR in HEK293 cells; 125I-AM radioligand binding assay; cAMP production assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro functional assay with mutagenesis and binding confirmation in a single rigorous study\",\n      \"pmids\": [\"11591721\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CL/RAMP3 forms a pharmacologically distinct adrenomedullin receptor (AM2 receptor) compared to CL/RAMP2 (AM1 receptor): CGRP8-37 effectively antagonizes AM at all species combinations of CL/RAMP3, while AM22-52 only antagonizes AM at the hCL/hRAMP3 combination, demonstrating species-dependent pharmacological discrimination between the two AM receptor subtypes.\",\n      \"method\": \"Transient expression in Cos7 cells; functional antagonism assays with AM22-52, CGRP8-37, and BIBN4096BS; apparent pA2 determination\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple species combinations tested across multiple receptor systems with quantitative pharmacology, replicated across endogenous cell lines\",\n      \"pmids\": [\"12970090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"N-glycosylation of RAMP3 at multiple consensus sites (2-4 of 4 sites used) and disulfide bond formation via all six conserved cysteine residues are required for functional CRLR/RAMP3 adrenomedullin receptor expression; elimination of all N-glycans inhibits 125I-AM binding and increases EC50 for AM, while elimination of all cysteines abolishes AM binding entirely.\",\n      \"method\": \"Xenopus oocyte expression system; 125I-AM radioligand binding; site-directed mutagenesis of glycosylation sites and cysteines; indirect evidence for disulfide bonds\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis, multiple orthogonal approaches in single study\",\n      \"pmids\": [\"12939163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Structure-function analysis using RAMP1-RAMP3 chimeras shows that helix 1 and residues 62-69 of helix 2 of RAMP1 are critical for CLR trafficking (association), while residues 86-89 at the junction of helix 3 and its connecting loop with helix 2 affect peptide potency but not CLR trafficking; the CLR recognition site is more exposed on RAMP1 than on RAMP3.\",\n      \"method\": \"RAMP1-RAMP3 chimera co-expression with CLR and CTR; CLR trafficking assay; agonist potency assays; molecular modeling\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — systematic chimera analysis with functional readouts, but single lab study\",\n      \"pmids\": [\"20017504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Glu74 in RAMP3 (equivalent to Trp74 in RAMP1) is important for AM binding and potency at the CLR/RAMP3 (AM2) receptor; Phe substitution at position 74 reduces AM potency in RAMP3 but not RAMP1, while Tyr has no effect in RAMP3 but enhances AM potency in RAMP1, indicating that geometry and charge of residue 74 differentially control AM interactions at AM2 vs. CGRP receptors.\",\n      \"method\": \"Site-directed mutagenesis of RAMP3 and RAMP1; co-expression with CLR in Cos7 cells; cAMP accumulation assay; ELISA for cell surface expression\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis with functional assay, single lab study\",\n      \"pmids\": [\"21402116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RAMP3 knockout mice are born normally (unlike RAMP2 KO which is embryonically lethal), but adult RAMP3-/- mice show delayed lymphatic drainage and impaired lymphatic endothelial cell migration in scratch-wound assays; AM administration fails to enhance re-endothelialization in RAMP3-/- cells, establishing a specific role for the AM-RAMP3 system in lymphatic vessel function and endothelial cell migration.\",\n      \"method\": \"RAMP3 knockout mice; lymphangiography with indocyanine green; 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 — KO mouse with multiple orthogonal phenotypic readouts and cell-level mechanistic follow-up\",\n      \"pmids\": [\"25264174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In rat mesenteric artery smooth muscle cells, RAMP3 (not RAMP2) associates with CALCRL to mediate ADM-induced cAMP production; RAMP3 knockdown inhibits ADM-induced cAMP production, and ADM treatment increases CALCRL-RAMP3 association as shown by proximity ligation assay.\",\n      \"method\": \"siRNA knockdown of RAMP2 or RAMP3; cAMP production assay; proximity ligation assay to detect CALCRL-RAMP3 interaction\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — knockdown with functional readout plus direct protein interaction assay, single lab\",\n      \"pmids\": [\"26423127\"],\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 via Rab4-positive vesicles following AM or SDF-1/CXCL12 binding; RAMP3 diminishes AM ligand availability without changing G-protein coupling; genetic deletion of RAMP3 in mice abolishes directed retinal angiogenesis.\",\n      \"method\": \"BRET-based interaction screen; cell-surface expression assays; Rab4-positive vesicle trafficking assay; RAMP3 knockout mice; retinal angiogenesis assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (BRET, trafficking, KO mouse phenotype) in single study, strong mechanistic follow-up\",\n      \"pmids\": [\"31712427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In the tumor microenvironment, RAMP3 mediates Src/Cas/podoplanin (PDPN) signaling in cancer-associated fibroblasts (CAFs); RAMP3 deficiency reduces PDPN-positive CAFs at metastatic sites, suppresses CAF migration and proliferation, and reduces liver metastasis of pancreatic cancer in mice.\",\n      \"method\": \"RAMP3 knockout mice; splenic injection of PAN02 pancreatic cancer cells for spontaneous liver metastasis; co-culture of CAFs and tumor cells; Src/Cas/PDPN signaling analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with in vivo metastasis readout and in vitro mechanistic pathway identification, single lab\",\n      \"pmids\": [\"31754214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RAMP3 KO mice show reduced anorectic response to amylin but retain response to salmon calcitonin, while RAMP1/3 double KO mice are insensitive to both, establishing that RAMP3 is required for part of amylin's food intake-suppressing effect; RAMP3 KO male mice are glucose intolerant, indicating a role in glucose homeostasis.\",\n      \"method\": \"RAMP1, RAMP3, and RAMP1/3 global KO mice; acute intraperitoneal amylin and salmon calcitonin injection; food intake measurement; glucose tolerance test\",\n      \"journal\": \"Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with pharmacological dissection, single lab\",\n      \"pmids\": [\"31881259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RAMP3 depletion in RAMP3 extracellular loops of the calcitonin receptor (CTR) fundamentally alters signal propagation through CTR ECL2 and ECL3 when forming the AMY3 receptor, with ligand-specific and pathway-specific (cAMP vs. ERK) effects of alanine mutations differing substantially from those in CTR alone.\",\n      \"method\": \"Alanine scanning mutagenesis of CTR ECL2 and ECL3 in presence of RAMP3; cAMP accumulation assay; ERK phosphorylation assay; expression in heterologous cells\",\n      \"journal\": \"ACS pharmacology & translational science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — systematic mutagenesis with two functional readouts, single lab\",\n      \"pmids\": [\"32219220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RAMP3 knockout mice show reduced systolic function and enhanced cardiac fibrosis after transverse aortic constriction (TAC), with a characteristic reduction in cardiac lymphatic vessels, establishing that the AM-RAMP3 system is required for later cardiac adaptation to stress through regulation of lymphatic vessels.\",\n      \"method\": \"Cardiomyocyte-specific and global RAMP3 KO mice; transverse aortic constriction (TAC); echocardiography; histological assessment of fibrosis and lymphatic vessels\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with multiple cardiac phenotype readouts, single lab\",\n      \"pmids\": [\"33545715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PTH induces RAMP3 mRNA expression as a primary response gene in osteoblasts primarily via the cAMP-PKA signaling pathway; this was demonstrated by the ability of forskolin (cAMP activator) to mimic PTH, H89 (PKA inhibitor) to block PTH- and FSK-induced RAMP3 expression, and insensitivity to the PKC-selective PTH fragment PTH(3-34).\",\n      \"method\": \"Primary mouse osteoblasts; RT-PCR for RAMP3 mRNA; pharmacological activation/inhibition of cAMP-PKA, PKC, and calcium pathways; cycloheximide chase to confirm primary response\",\n      \"journal\": \"Calcified tissue international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological pathway dissection with multiple agonists/inhibitors, single lab\",\n      \"pmids\": [\"16075364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RAMP3 is an early target gene of the canonical Wnt/β-catenin pathway, with RAMP3 mRNA upregulated within 1-6 hours of Wnt-3a stimulation in C57MG cells.\",\n      \"method\": \"Wnt-3a stimulation of C57MG cells; time-course RT-PCR/mRNA expression analysis\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single method (expression analysis), no direct mechanistic follow-up of RAMP3 function downstream of Wnt\",\n      \"pmids\": [\"19353769\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RAMP3 binds ACKR2 in hepatocellular carcinoma tumor cells and promotes membrane distribution of ACKR2 via RAB4-positive vesicles, thereby facilitating CCL2 scavenging/degradation; RAMP3 expression inhibits CD11b+/Gr-1+ myeloid cell infiltration and neovascularization in tumors through ACKR2.\",\n      \"method\": \"Co-immunoprecipitation/protein-protein interaction assay; RAB4 vesicle trafficking assay; CCL2 scavenging assay; syngeneic mouse tumor model with Hepa1-6 cells; flow cytometry for myeloid cells\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional assays in vitro and in vivo with mechanistic pathway identification, single lab\",\n      \"pmids\": [\"39437486\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RAMP3 interacts with GLP-1R and biases its signaling away from canonical cAMP toward Ca2+ mobilization and increased Gαq/Gαi coupling at the expense of Gαs; this interaction enhances glucose-stimulated insulin secretion in cells overexpressing RAMP3, and RAMP3 KO mice show reduced sensitivity to GLP-1 in glucose and insulin tolerance tests.\",\n      \"method\": \"BRET-based interaction assay; cAMP assay; Ca2+ mobilization assay; G protein coupling assays; glucose-stimulated insulin secretion assay; RAMP3 KO mouse glucose/insulin tolerance tests\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal signaling assays plus in vivo KO validation, 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\": \"AMY3R (CTR/RAMP3 heterodimer) has a stronger transmembrane domain interface than AMY1/2R, yielding a more stable heterodimer; human and salmon calcitonin agonists promote AMY3R dissociation, while amylin and αCGRP promote association of AMY1/2R, demonstrating that agonist-modulated subunit interaction dynamics control cAMP signaling output.\",\n      \"method\": \"Biochemical assay resolving AMYR heterodimers from free subunits; live cell membrane subunit association assays; G protein coupling assays; cAMP signaling assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — novel biochemical assay plus multiple functional 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 by forming heterodimeric complexes with multiple GPCRs — most notably CLR (to form the AM2/adrenomedullin receptor), CTR (to form the AMY3 amylin receptor), ACKR3 (regulating chemokine scavenging and receptor recycling via Rab4-positive vesicles), ACKR2 (promoting CCL2 degradation), and GLP-1R (biasing signaling toward Ca2+/Gαq/Gαi and enhancing insulin secretion) — with RAMP3's extracellular N-terminal domain (including conserved cysteines forming disulfide bonds, N-glycosylation sites, and specific residues at positions 59-65 and 74) critical for ligand-binding specificity, while in vivo RAMP3 is dispensable for vascular development but required for lymphatic drainage, retinal angiogenesis, cardiac adaptation to stress, and metabolic regulation of food intake and glucose homeostasis.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"RAMP3, together with RAMP1 and RAMP2, forms a family of single-transmembrane-domain proteins required to transport the calcitonin receptor-like receptor (CRLR) to the plasma membrane; RAMP2- or RAMP3-transported CRLR functions as an adrenomedullin receptor, whereas RAMP1-transported CRLR functions as a CGRP receptor.\",\n      \"method\": \"Transfection/co-expression in cell lines with radioligand binding and functional cAMP assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — foundational discovery paper replicated widely across labs, multiple orthogonal methods\",\n      \"pmids\": [\"9620797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"RAMP3 co-expressed with the calcitonin receptor (CTR) generates a distinct amylin receptor phenotype (AMY3R) with reduced efficacy for human CGRPα compared to the RAMP1/CTR (AMY1R) combination; RAMP3 is located intracellularly when expressed alone but traffics to the cell surface upon co-expression with CTR.\",\n      \"method\": \"Co-transfection in COS-7 cells, 125I-amylin radioligand binding competition, cAMP functional assays, confocal microscopy with c-myc-tagged RAMP1, and cross-linking experiments\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"10385705\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CRLR is endocytosed together with RAMP3 (and RAMP1/2) via clathrin-coated vesicles following agonist stimulation; internalized CRLR/RAMP complexes are largely targeted to a degradative (lysosomal) pathway with inefficient recycling, and CRLR cannot reach the plasma membrane without RAMPs.\",\n      \"method\": \"Stable expression of CRLR-GFP chimera ± RAMPs in HEK293 cells; agonist-stimulated internalization tracked by fluorescence microscopy; hypertonic sucrose inhibition of clathrin-dependent endocytosis; co-localization with rhodamine-transferrin and lysosomal markers\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — live-cell imaging with functional validation and pharmacological controls\",\n      \"pmids\": [\"10882736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Direct protein–protein interaction between RAMP3 (or RAMP2) and CRLR, not differential glycosylation, determines ligand-binding selectivity: cross-linking experiments showed that 125I-AM is incorporated into RAMP2 and RAMP3 within the CRLR-RAMP heterodimer, placing the RAMPs close to the peptide-binding pocket; fully glycosylated CRLR, not the core-glycosylated form produced by RAMP2/3 co-expression, represents the functional AM-binding receptor.\",\n      \"method\": \"Co-expression in HEK293 cells, cross-linking with bis-succinimidyl suberate, radioligand binding (125I-AM, 125I-CGRP), glycosylation analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — biochemical reconstitution with cross-linking and radioligand binding, multiple orthogonal methods\",\n      \"pmids\": [\"11387328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Seven specific amino acids in the extracellular N-terminal domain of RAMP3 (residues 59–65) are critical for high-affinity AM binding and AM-evoked cAMP production at the CRLR/RAMP3 adrenomedullin receptor; deletion of this segment significantly attenuates 125I-AM binding despite preserved cell-surface expression of the receptor heterodimer.\",\n      \"method\": \"RAMP chimeras and deletion mutants co-expressed with CRLR in HEK293 cells; 125I-AM radioligand binding; AM-evoked cAMP production assay; cell-surface expression confirmed by ELISA\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis combined with radioligand binding and functional cAMP assay\",\n      \"pmids\": [\"11591721\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"RAMP3 interacts with the PTH2 receptor (in addition to CRLR and CTR), extending the known repertoire of RAMP3 receptor partners to at least one additional class II GPCR; RAMP-receptor complexes beyond CL/CTR can modulate downstream signaling.\",\n      \"method\": \"Immunofluorescence confocal microscopy co-localization; radioligand binding in COS-7 cells; phosphoinositide hydrolysis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single-lab co-localization study with partial functional follow-up\",\n      \"pmids\": [\"12446722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"N-glycosylation of RAMP3 (at two to four of its four consensus sites) and disulfide bonds formed by all six conserved cysteine residues in its extracellular domain are each essential for functional expression of the CRLR/RAMP3 adrenomedullin receptor: elimination of all N-glycans reduces 125I-AM binding and increases EC50, while mutation of all cysteines to serine completely abolishes AM binding.\",\n      \"method\": \"Xenopus oocyte expression system; site-directed mutagenesis of N-glycosylation sites and cysteines; 125I-AM radioligand binding; EC50 determination for AM-evoked responses\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis with radioligand binding and functional assay in a well-controlled expression system\",\n      \"pmids\": [\"12939163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CL/RAMP2 and CL/RAMP3 receptors are pharmacologically distinct: AM22-52 selectively antagonizes CL/RAMP2 over CL/RAMP3 in human receptor combinations, whereas CGRP8-37 is an effective antagonist at CL/RAMP3 but shows species-dependent effects at CL/RAMP2; the agonist potency order at CL/RAMP3 is AM ≈ βCGRP > αCGRP.\",\n      \"method\": \"Transient transfection in COS-7 cells and endogenous receptor expression in Rat 2 and L6 cells; cAMP functional assays with peptide antagonists; apparent pA2 determination\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological characterization across multiple receptor combinations and cell systems\",\n      \"pmids\": [\"12970090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The C-terminal PDZ type I motif unique to RAMP3 (absent from RAMP1 and RAMP2) mediates interaction with N-ethylmaleimide-sensitive factor (NSF), redirecting the CRLR/RAMP3 (AM2R) complex from a degradative pathway to a recycling pathway after agonist-stimulated internalization; deletion or point mutation of the PDZ motif abolishes this NSF interaction and recycling.\",\n      \"method\": \"Adenylate cyclase assays, radioligand binding, immunofluorescence microscopy in HEK293 cells; mutational analysis (deletion and point mutations of PDZ motif); RNA interference of RAMP3 in rat mesangial cells; pharmacological inhibition of NSF\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis + RNAi + pharmacological inhibition with multiple functional readouts, replicated in endogenous cell system\",\n      \"pmids\": [\"15613468\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Parathyroid hormone (PTH) induces RAMP3 mRNA expression in primary mouse osteoblasts as a primary response gene (cycloheximide-insensitive) primarily through the cAMP–PKA signaling pathway; PKA inhibition with H89 blocks PTH- and forskolin-induced RAMP3 upregulation, whereas PKC activation with PMA also increases RAMP3 but calcium signaling (ionomycin) does not.\",\n      \"method\": \"Primary mouse osteoblast cultures; representational difference analysis; RT-PCR for RAMP3 mRNA; pharmacological dissection using cycloheximide, forskolin, H89 (PKA inhibitor), PMA (PKC activator/depletor), ionomycin, and PTH(3-34)\",\n      \"journal\": \"Calcified tissue international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — pharmacological pathway dissection in primary cells, single lab\",\n      \"pmids\": [\"16075364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Residue 74 in RAMP3 (Glu74) is a key determinant of AM pharmacology at the CL/RAMP3 (AM2) receptor; substitution of RAMP3 residues into RAMP1 and vice versa at position 74 converts AM potency profiles, with RAMP1 Phe93 additionally contributing to αCGRP potency at the CGRP receptor.\",\n      \"method\": \"Site-directed mutagenesis of RAMP1 and RAMP3 at eight conserved positions; co-transfection with CL or CTR in COS-7 cells; agonist-stimulated cAMP production; cell-surface expression by ELISA\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — systematic mutagenesis with functional and expression readouts\",\n      \"pmids\": [\"18593822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RAMP3 is an early Wnt target gene: in C57MG cells stimulated with Wnt-3a, RAMP3 mRNA is induced within 1–6 hours, classifying it as an early (not immediate or late) transcriptional target of canonical Wnt/β-catenin signaling.\",\n      \"method\": \"Wnt-3a stimulation of C57MG cells; time-course RT-PCR for RAMP3 and other Wnt target genes; comparison of induction kinetics\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single-lab expression kinetics study; mechanistic implication (Wnt regulation of RAMP3) supported by pathway context\",\n      \"pmids\": [\"19353769\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Structure–function analysis of RAMP1–RAMP3 chimeras reveals that extracellular helix 1 and residues 62–69 of helix 2 are required for CRLR trafficking (CLR association), while residues 86–89 at the helix 2/3 junction influence peptide (αCGRP and AM) binding potency without affecting trafficking; RAMP3 chimera data suggest the helix 2/3 loop is less exposed than in RAMP1 and plays a role in peptide binding.\",\n      \"method\": \"RAMP1/RAMP3 chimera co-expression with CLR or CTR in COS-7 cells; CLR trafficking assay; agonist-stimulated cAMP production; homology modeling\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — chimeric mutagenesis with trafficking and functional readouts, modeling provides mechanistic context\",\n      \"pmids\": [\"20017504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Glu74 in RAMP3 specifically contributes to AM potency at the AM2 (CL/RAMP3) receptor through geometry and charge: Phe substitution reduces AM potency while Tyr does not, and most other substitutions have small effects; the equivalent residue Trp74 in RAMP1 has distinct behavior (Tyr enhances AM potency), confirming position 74 as a key differential determinant between AM2 and CGRP receptors.\",\n      \"method\": \"Site-directed mutagenesis of Glu74 in RAMP3 and Trp74 in RAMP1 with multiple amino acid substitutions; co-expression with CLR in COS-7 cells; cAMP assay for AM, AM2/intermedin, and CGRP; cell-surface expression by ELISA\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — systematic mutagenesis with functional and expression readouts confirming prior findings\",\n      \"pmids\": [\"21402116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RAMP3 physically interacts with GPR30 (G-protein-coupled estrogen receptor 1/GPER1): BRET titration, co-immunoprecipitation, and confocal microscopy demonstrate direct interaction; GPR30 presence increases RAMP3 plasma membrane expression in HEK293 cells; in vivo, RAMP3 knockout disrupts GPR30 subcellular localization in cardiac cells and abolishes GPR30-agonist (G-1)-mediated reduction of cardiac hypertrophy and perivascular fibrosis in a sex-dependent manner.\",\n      \"method\": \"BRET titration assays, co-immunoprecipitation, confocal microscopy in HEK293 cells; in vivo RAMP3 knockout mice on heart disease-prone background treated with G-1; cardiac histopathology\",\n      \"journal\": \"Journal of molecular endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal in vitro methods plus in vivo validation with defined phenotypic readout\",\n      \"pmids\": [\"23674134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RAMP3 knockout mice are viable and display normal postnatal blood vessel angiogenesis, but exhibit delayed lymphatic drainage (shown by ICG lymphangiography), delayed chyle transport by intestinal lymphatics, more severe interstitial edema in a tail lymphedema model, and a primary migration defect in isolated RAMP3−/− lymphatic endothelial cells that is not rescued by AM; RAMP2 knockout (but not RAMP3 knockout) is embryonically lethal due to vascular defects, establishing distinct functional roles for the two AM receptor subtypes.\",\n      \"method\": \"RAMP2−/− and RAMP3−/− mouse generation; ICG lymphangiography; tail lymphedema model with histology; in vitro scratch-wound migration assay of isolated lymphatic endothelial cells\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with multiple orthogonal in vivo and in vitro phenotypic readouts\",\n      \"pmids\": [\"25264174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In rat mesenteric artery smooth muscle cells (VSMCs), RAMP3 (not RAMP2) associates with CALCRL to mediate ADM-induced cAMP production; RAMP3 knockdown inhibits ADM-induced cAMP generation and ADM–CALCRL association; proximity ligation assay directly demonstrates RAMP3–CALCRL co-localization enhanced by ADM.\",\n      \"method\": \"RAMP2/RAMP3 siRNA knockdown in VSMCs; cAMP production assay with peptide antagonists; proximity ligation assay for RAMP3–CALCRL association\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNAi knockdown and proximity ligation in primary-like cells, single lab\",\n      \"pmids\": [\"26423127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RAMP3 interacts 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; RAMP3–ACKR3 interaction reduces AM ligand availability without changing G-protein coupling; genetic deletion of RAMP3 in mice abolishes directed retinal angiogenesis, phenocopying ACKR3 deletion.\",\n      \"method\": \"BRET-based and cell-surface expression screens for RAMP–chemokine receptor interactions; Rab4-positive vesicle trafficking assays; AM ligand scavenging assays; RAMP3 and ACKR3 global knockout mice; retinal angiogenesis imaging\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — comprehensive BRET screen, mechanistic trafficking assays, and in vivo genetic validation with defined phenotype\",\n      \"pmids\": [\"31712427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RAMP3 deficiency in cancer-associated fibroblasts (CAFs) suppresses tumor metastasis by reducing podoplanin (PDPN)-positive CAF numbers at metastatic sites; RAMP3 regulates PDPN expression via Src/Cas/PDPN signaling in fibroblasts; RAMP3−/− CAFs show reduced migration, proliferation, and metastasis-promoting activity in co-cultures with tumor cells in vitro and in vivo.\",\n      \"method\": \"Splenic injection of PAN02 pancreatic cancer cells in RAMP3−/− and DI-E-RAMP2−/− mice for spontaneous liver metastasis model; podoplanin immunohistochemistry; Src/Cas pathway analysis; in vitro co-culture migration and proliferation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO model with mechanistic pathway analysis (Src/Cas/PDPN) and in vitro corroboration, single lab\",\n      \"pmids\": [\"31754214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RAMP3 knockout mice display glucose intolerance (on chow diet in males) and altered meal patterns (decreased intermeal interval); amylin and salmon calcitonin anorectic effects are mediated through distinct RAMP-containing receptor combinations: RAMP1/3 double KO mice are insensitive to both amylin and sCT, while RAMP3 KO mice respond to sCT but not amylin, indicating RAMP3 specifically mediates part of amylin's anorectic signaling.\",\n      \"method\": \"Global RAMP1, RAMP3, and RAMP1/3 double KO mice; glucose tolerance tests; intraperitoneal amylin and sCT injection with food intake measurement; meal pattern analysis; c-Fos immunohistochemistry in area postrema\",\n      \"journal\": \"Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with multiple metabolic readouts, single lab\",\n      \"pmids\": [\"31881259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RAMP3 alanine scanning of extracellular loops (ECL2 and ECL3) of the calcitonin receptor in the AMY3R context demonstrates that RAMP3 co-expression fundamentally alters the dynamic role of ECL2 and ECL3 in propagating cAMP and ERK signaling compared to CTR alone; the effects are highly peptide-dependent (sCT, hCT, pCT, rat amylin, hCGRP show distinct ECL mutation effects in the AMY3R context).\",\n      \"method\": \"Alanine scanning mutagenesis of CTR ECL2 and ECL3 co-expressed with RAMP3; cAMP accumulation assay; ERK phosphorylation (pERK) assay; cell-surface expression by ELISA\",\n      \"journal\": \"ACS pharmacology & translational science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — systematic mutagenesis with two signaling readouts, single lab\",\n      \"pmids\": [\"32219220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cryo-EM structures of the AM2R (CL/RAMP3) in complex with AM or intermedin and Gs protein reveal that RAMP3 positions its extracellular domain (ECD) at a distinct primary orientation relative to the receptor core compared to RAMP2 in AM1R, and that the RAMP linker region connecting the TM helix and ECD controls receptor phenotype; distinct ECL3 positioning and ECD mobility are receptor-dependent features.\",\n      \"method\": \"Cryo-EM structure determination of AM2R–AM–Gs (2.4 Å) and AM2R–AM2–Gs (2.3 Å); chimeric RAMP linker exchange experiments; dynamic analysis of cryo-EM micrographs\",\n      \"journal\": \"ACS pharmacology & translational science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution cryo-EM structures with chimeric functional validation\",\n      \"pmids\": [\"32296767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cardiomyocyte-specific RAMP3 knockout mice show normal baseline cardiac function but develop reduced systolic function and enhanced fibrosis after 4 weeks of transverse aortic constriction (TAC), with a characteristic reduction in cardiac lymphatic vessels, indicating that the AM–RAMP3 system is required for later adaptive cardiac responses to cardiovascular stress through regulation of lymphatic vessels.\",\n      \"method\": \"Cardiomyocyte-specific RAMP3 KO mice; TAC model; echocardiography; histological fibrosis quantification; cardiac lymphatic vessel immunostaining\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — tissue-specific KO with defined phenotypic readout, single lab\",\n      \"pmids\": [\"33545715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM structures of AMY3R (CTR/RAMP3) with amylin and other calcitonin family peptides demonstrate that RAMP3 constrains the conformation of amylin-bound AMY3R through a midpeptide 'bypass motif', and that CT-based peptide activation of AMY3R is structurally distinct from amylin-based activation; these structural differences have direct implications for ligand selectivity.\",\n      \"method\": \"Cryo-EM structure determination of AMY3R–amylin, AMY1R–sCT, AMY2R–sCT/hCT, and CTR–amylin/sCT/hCT complexes; structural comparison and dynamics analysis\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution cryo-EM structures of multiple complexes with rigorous comparative analysis\",\n      \"pmids\": [\"35324283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RAMP3 binds atypical chemokine receptor 2 (ACKR2) in HCC tumor cells and promotes ACKR2 membrane distribution through Rab4-positive vesicles, enabling ACKR2-mediated CCL2 scavenging/degradation; RAMP3 expression reduces intratumoral CCL2, inhibits STAT3 and AKT phosphorylation, and reduces CD11b+/Gr-1+ myeloid cell infiltration and neovascularization in syngeneic tumors through ACKR2.\",\n      \"method\": \"Co-IP and bioinformatic PPI analysis; overexpression and knockdown in Huh7, HepG2, and Hepa1-6 cells; syngeneic tumor implantation; CCL2 ELISA; STAT3/AKT phosphorylation Western blot; flow cytometry for myeloid infiltration; immunohistochemistry for neovascularization\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple in vitro and in vivo methods, single lab, extends ACKR interaction repertoire of RAMP3\",\n      \"pmids\": [\"39437486\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RAMP3 interacts with the GLP-1 receptor (GLP-1R) and biases its signaling: RAMP3 co-expression reduces canonical Gαs/cAMP coupling while increasing Gαq and Gαi coupling, shifting the receptor toward Ca2+ mobilization; this altered signaling profile elevates glucose-stimulated insulin secretion in cells overexpressing RAMP3, and RAMP3 knockout mice show reduced sensitivity to GLP-1 in glucose and insulin tolerance tests.\",\n      \"method\": \"BRET assays for GLP-1R–RAMP3 interaction and G-protein coupling; Ca2+ mobilization assay; cAMP assay; glucose-stimulated insulin secretion assay; RAMP3 KO mouse glucose and insulin tolerance tests\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal signaling assays plus in vivo KO validation, but 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\": \"AMY3R (CTR/RAMP3) has a stronger transmembrane domain interface than AMY1R or AMY2R, resulting in a more stable basal CTR–RAMP3 subunit association; calcitonin agonists (human and salmon CT) promote AMY3R dissociation, while rat amylin promotes AMY1/2R subunit association; these altered equilibria directly affect G-protein coupling and cAMP signaling output.\",\n      \"method\": \"Novel biochemical assay resolving AMYR heterodimers vs. free subunits; live-cell membrane subunit association assays; G-protein coupling assays; cAMP signaling assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — novel biochemical approach with multiple functional readouts, but 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 heterodimerizes with multiple GPCRs (primarily CLR and CTR, but also ACKR3, ACKR2, GPR30, PTH2R, and GLP-1R) to act as a molecular chaperone promoting cell-surface expression, a determinant of ligand-binding pharmacology through its extracellular N-terminal domain (particularly residues 59–65 and Glu74), and a regulator of post-endocytic receptor trafficking via its unique C-terminal PDZ motif that recruits NSF to redirect internalized receptors toward recycling rather than degradation; in vivo, RAMP3 is required for lymphatic vessel function, guided angiogenesis through ACKR3-dependent chemotactic gradient formation, sex-dependent GPR30-mediated cardioprotection, glucose homeostasis, and amylin-mediated satiation signaling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RAMP3 is a single-pass transmembrane accessory protein that heterodimerizes with multiple GPCRs to modulate their trafficking, ligand specificity, and signaling bias. It partners with CLR to form the AM2 adrenomedullin receptor, where its extracellular N-terminal domain—including residues 59–65, Glu74, conserved disulfide-forming cysteines, and N-glycosylation sites—determines high-affinity AM binding and cAMP production [PMID:11591721, PMID:12939163, PMID:21402116]. RAMP3 also complexes with CTR to form the AMY3 amylin receptor that allosterically reshapes extracellular loop signaling [PMID:32219220], and with ACKR3 and ACKR2 to promote Rab4-dependent receptor recycling and chemokine scavenging [PMID:31712427, PMID:39437486]. In vivo, RAMP3 is dispensable for vascular development but is required for lymphatic drainage, retinal angiogenesis, cardiac adaptation to pressure overload, and metabolic regulation of food intake and glucose homeostasis [PMID:25264174, PMID:31712427, PMID:33545715, PMID:31881259].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Identifying the ligand-binding determinants of RAMP3 established that a discrete seven-residue extracellular segment (59–65) confers high-affinity AM binding to the CLR/RAMP3 heterodimer without affecting receptor surface expression.\",\n      \"evidence\": \"Chimeric and deletion RAMP constructs co-expressed with CRLR in HEK293 cells; 125I-AM binding and cAMP assays\",\n      \"pmids\": [\"11591721\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic contacts between RAMP3 residues 59–65 and AM undefined\", \"Role of individual residues within this stretch not resolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstrating species-dependent antagonist profiles at CLR/RAMP3 versus CLR/RAMP2 established that RAMP3 creates a pharmacologically distinct AM2 receptor subtype, while post-translational requirements (N-glycosylation, disulfide bonds) were shown to be essential for functional receptor expression.\",\n      \"evidence\": \"Cos7 cell pharmacology with cross-species CLR/RAMP combinations; Xenopus oocyte mutagenesis of glycosylation sites and cysteines\",\n      \"pmids\": [\"12970090\", \"12939163\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crystal/cryo-EM structure of CLR/RAMP3 ectodomain complex not yet available at this time\", \"Relative contribution of individual glycosylation sites to folding vs. ligand contact unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Systematic RAMP1–RAMP3 chimera analysis resolved that distinct helical segments control two separable functions—CLR trafficking (helix 1 and helix 2 residues 62–69) versus peptide potency (helix 3/loop junction residues 86–89)—and revealed that the CLR recognition site is more exposed on RAMP1 than RAMP3.\",\n      \"evidence\": \"RAMP1-RAMP3 chimera co-expression with CLR and CTR; trafficking and agonist potency assays; molecular modeling\",\n      \"pmids\": [\"20017504\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Only chimeras tested; point-mutation validation of individual residues incomplete\", \"Structural basis for differential CLR exposure across RAMPs unresolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identifying Glu74 as a specificity-determining residue in RAMP3 showed that a single position differentially controls AM potency at the AM2 versus CGRP receptor, advancing understanding of how RAMP identity dictates ligand selectivity.\",\n      \"evidence\": \"Site-directed mutagenesis at position 74 in RAMP3 and RAMP1; cAMP assays in Cos7 cells\",\n      \"pmids\": [\"21402116\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct biophysical measurement of Glu74–AM contact\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showing that RAMP3 knockout mice are viable (unlike RAMP2 KO) but display impaired lymphatic drainage and endothelial migration established a non-redundant in vivo role for the AM–RAMP3 axis in lymphatic vessel function.\",\n      \"evidence\": \"RAMP3 KO mice; lymphangiography; tail lymphedema model; scratch-wound assay with primary lymphatic endothelial cells\",\n      \"pmids\": [\"25264174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream intracellular signaling pathway linking RAMP3 to lymphatic migration not identified\", \"Contribution of RAMP3 partners other than CLR to the lymphatic phenotype not excluded\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Discovery that RAMP3 heterodimerizes with ACKR3 (an atypical chemokine receptor) and directs its recycling through Rab4-positive vesicles broadened RAMP3's role beyond classical GPCRs and linked it to retinal angiogenesis in vivo.\",\n      \"evidence\": \"BRET interaction screen; cell-surface expression and Rab4 vesicle trafficking assays; RAMP3 KO mouse retinal angiogenesis\",\n      \"pmids\": [\"31712427\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for RAMP3–ACKR3 interaction unknown\", \"Whether RAMP3 similarly regulates other atypical chemokine receptors untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Alanine-scanning of CTR extracellular loops in the presence versus absence of RAMP3 revealed that RAMP3 allosterically reorganizes signal propagation through CTR (AMY3R), with pathway-specific (cAMP vs. ERK) and ligand-specific effects.\",\n      \"evidence\": \"Systematic alanine mutagenesis of CTR ECL2/ECL3 ± RAMP3; cAMP and ERK phosphorylation assays in heterologous cells\",\n      \"pmids\": [\"32219220\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural visualization of RAMP3-induced conformational change in CTR ECLs\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrating that RAMP3 KO mice have blunted anorectic responses to amylin and impaired glucose tolerance established physiological roles for RAMP3 in energy and glucose homeostasis, likely through the AMY3 amylin receptor.\",\n      \"evidence\": \"RAMP1, RAMP3, and RAMP1/3 global KO mice; acute amylin and salmon calcitonin injection; food intake and glucose tolerance tests\",\n      \"pmids\": [\"31881259\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Brain region and cell type mediating RAMP3-dependent amylin action not identified\", \"Relative contribution of AMY3R versus other RAMP3 complexes to glucose phenotype unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showing that RAMP3 KO mice develop worse cardiac fibrosis and reduced lymphatic vessels after pressure overload extended the lymphatic role of RAMP3 to cardiac adaptation to stress.\",\n      \"evidence\": \"Cardiomyocyte-specific and global RAMP3 KO mice; transverse aortic constriction; echocardiography; histology\",\n      \"pmids\": [\"33545715\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cardiomyocyte-intrinsic versus lymphatic endothelial contribution not fully resolved\", \"Molecular mechanism linking RAMP3 to cardiac lymphangiogenesis not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identification of RAMP3 as a binding partner of ACKR2 that promotes its membrane distribution via Rab4 vesicles and facilitates CCL2 scavenging expanded the RAMP3–atypical chemokine receptor axis to immune modulation in tumors.\",\n      \"evidence\": \"Co-immunoprecipitation; Rab4 trafficking assay; CCL2 scavenging assay; syngeneic mouse HCC model\",\n      \"pmids\": [\"39437486\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RAMP3 directly contacts ACKR2 or acts through intermediaries not resolved\", \"Single-lab study with one tumor model\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"No high-resolution structure of a full-length RAMP3–GPCR complex exists, and the mechanism by which RAMP3 directs receptors to Rab4-positive recycling pathways remains undefined at the molecular level.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for RAMP3 selectivity among GPCRs unknown\", \"Molecular determinants of RAMP3-mediated Rab4 sorting not identified\", \"Signaling bias mechanisms for RAMP3 interactions with non-CLR partners lack structural explanation\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 3, 7, 10, 14]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 4, 10, 7, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 6, 7, 14]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [7, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0009609507\", \"supporting_discovery_ids\": [7, 14]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 6, 10, 9]}\n    ],\n    \"complexes\": [\n      \"CLR/RAMP3 (AM2 receptor)\",\n      \"CTR/RAMP3 (AMY3 receptor)\",\n      \"ACKR3/RAMP3\",\n      \"ACKR2/RAMP3\"\n    ],\n    \"partners\": [\n      \"CALCRL\",\n      \"CALCR\",\n      \"ACKR3\",\n      \"ACKR2\",\n      \"RAB4\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"RAMP3 is a single-transmembrane accessory protein that heterodimerizes with multiple GPCRs to regulate their trafficking, pharmacology, and signaling. It was originally identified as a chaperone that escorts the calcitonin receptor-like receptor (CLR) to the cell surface, where CLR/RAMP3 forms the AM2 adrenomedullin receptor, with residues 59–65 and Glu74 in the RAMP3 extracellular domain determining high-affinity AM binding [PMID:9620797, PMID:11591721, PMID:18593822]. A unique C-terminal PDZ motif distinguishes RAMP3 from RAMP1/RAMP2 by recruiting NSF to redirect internalized receptor complexes from lysosomal degradation to Rab4-positive recycling endosomes, a mechanism operative for both CLR and the atypical chemokine receptors ACKR3 and ACKR2 [PMID:15613468, PMID:31712427, PMID:39437486]. In vivo, RAMP3 is required for lymphatic vessel function, ACKR3-dependent retinal angiogenesis, amylin-mediated satiation signaling, GPR30-dependent cardioprotection, and glucose homeostasis [PMID:25264174, PMID:31712427, PMID:31881259, PMID:23674134].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Identification of RAMP3 as part of a new family of single-TM accessory proteins required for CRLR plasma membrane delivery and ligand-specificity switching (CGRP vs. AM receptor) resolved the longstanding paradox of how a single receptor generated two pharmacologically distinct binding phenotypes.\",\n      \"evidence\": \"Co-expression in cell lines with radioligand binding and cAMP assays\",\n      \"pmids\": [\"9620797\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and direct physical interaction not yet demonstrated\", \"Mechanism of selectivity switch unknown\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Demonstration that RAMP3 also partners with the calcitonin receptor to generate a distinct amylin receptor (AMY3R) established that RAMPs are not CRLR-specific accessories but general GPCR modulators, and showed RAMP3 requires a receptor partner for surface expression.\",\n      \"evidence\": \"Co-transfection in COS-7 cells with radioligand binding, cAMP assays, and confocal microscopy\",\n      \"pmids\": [\"10385705\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full repertoire of RAMP3-interacting GPCRs unknown\", \"Structural basis of AMY3R vs. AMY1R selectivity unresolved\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Cross-linking and mutagenesis studies established that RAMP3 directly contacts the ligand-binding pocket and that residues 59–65 in its extracellular domain are critical for AM binding, shifting the mechanistic model from glycosylation-dependent to direct protein–protein interaction-dependent pharmacology.\",\n      \"evidence\": \"Cross-linking with 125I-AM, RAMP chimera/deletion mutagenesis with radioligand binding and cAMP assays in HEK293 cells\",\n      \"pmids\": [\"11387328\", \"11591721\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of the RAMP3–CLR interface not available\", \"Contribution of individual residues within 59–65 not dissected\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Post-translational requirements for RAMP3 function were defined: N-glycosylation and disulfide bonds formed by all six conserved cysteines are essential for functional AM receptor expression, and pharmacological profiling established that AM2R (CLR/RAMP3) has distinct antagonist sensitivity from AM1R (CLR/RAMP2).\",\n      \"evidence\": \"Mutagenesis in Xenopus oocytes and pharmacological characterization in COS-7 cells with cAMP assays\",\n      \"pmids\": [\"12939163\", \"12970090\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of RAMP3 ECD not solved\", \"Species-dependent pharmacological differences not fully explained mechanistically\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Discovery that the C-terminal PDZ motif unique to RAMP3 recruits NSF to reroute internalized CLR/RAMP3 from degradation to recycling answered why AM2R resensitizes more efficiently than AM1R and introduced a trafficking-based mechanism for differential RAMP function.\",\n      \"evidence\": \"Mutagenesis, RNAi, and pharmacological NSF inhibition in HEK293 and rat mesangial cells with cAMP, binding, and immunofluorescence readouts\",\n      \"pmids\": [\"15613468\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of PDZ domain-containing scaffold linking RAMP3 to NSF not determined\", \"Whether recycling mechanism applies to non-CLR partners unknown at this point\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Systematic mutagenesis pinpointed Glu74 as the single residue most critical for differential AM potency between AM2R and CGRP receptor, providing the first residue-level explanation for RAMP-dependent pharmacological switching.\",\n      \"evidence\": \"Site-directed mutagenesis of eight conserved positions in RAMP1/RAMP3 with cAMP and surface expression assays\",\n      \"pmids\": [\"18593822\", \"21402116\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural context of Glu74 within the peptide-binding interface not visualized\", \"Role in intermedin/AM2 binding not fully assessed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"RAMP3 was shown to physically interact with GPR30 (GPER1), a non-class-B GPCR, expanding the interactome well beyond CLR/CTR; in vivo, RAMP3 deletion abolished GPR30-agonist-mediated cardioprotection in a sex-dependent manner.\",\n      \"evidence\": \"BRET, co-IP, confocal in HEK293; RAMP3 KO mice with TAC and G-1 treatment; cardiac histopathology\",\n      \"pmids\": [\"23674134\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which RAMP3 modulates GPR30 signaling (trafficking vs. coupling) not dissected\", \"Sex-dependence mechanism unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"RAMP3 knockout mice revealed a non-redundant role for RAMP3 in lymphatic vessel function — lymphatic drainage, chyle transport, and lymphatic endothelial cell migration were all impaired — distinguishing RAMP3 from RAMP2 whose loss causes embryonic-lethal vascular defects.\",\n      \"evidence\": \"Global RAMP3 KO mice; ICG lymphangiography; tail lymphedema model; isolated lymphatic EC migration assay\",\n      \"pmids\": [\"25264174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which RAMP3 receptor partner mediates lymphatic effects not identified\", \"Whether PDZ-dependent recycling is involved in lymphatic function untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Three independent 2019 studies collectively broadened RAMP3 biology: (1) RAMP3 interaction with ACKR3 enables Rab4-dependent receptor recycling and AM scavenging required for retinal angiogenesis; (2) RAMP3 in cancer-associated fibroblasts regulates podoplanin via Src/Cas signaling to promote metastasis; (3) RAMP3 KO mice are glucose intolerant and lose amylin-mediated anorexia, establishing RAMP3 in metabolic homeostasis.\",\n      \"evidence\": \"BRET screens, Rab4 trafficking assays, retinal angiogenesis in KO mice; syngeneic tumor metastasis models with Src/Cas pathway analysis; metabolic phenotyping of global RAMP1/RAMP3 KO mice with amylin/sCT injection\",\n      \"pmids\": [\"31712427\", \"31754214\", \"31881259\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RAMP3 PDZ motif mediates ACKR3 recycling specifically through NSF not shown\", \"CAF findings from single cancer model\", \"Brain region-specific RAMP3 function in satiation not mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Cryo-EM structures of AM2R (CLR/RAMP3) at 2.3–2.4 Å resolution revealed how RAMP3 ECD orientation and the linker region between TM and ECD control receptor phenotype, providing the first atomic-level explanation for RAMP-dependent pharmacology.\",\n      \"evidence\": \"Cryo-EM of AM2R–AM–Gs and AM2R–intermedin–Gs; chimeric RAMP linker swaps\",\n      \"pmids\": [\"32296767\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of RAMP3 in complex with CTR (AMY3R) at this time\", \"Intracellular PDZ domain region not resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Cryo-EM of AMY3R (CTR/RAMP3) showed that RAMP3 constrains amylin binding through a midpeptide bypass motif and that calcitonin-based versus amylin-based activation proceeds through structurally distinct mechanisms, explaining peptide-dependent signaling differences.\",\n      \"evidence\": \"Cryo-EM structures of AMY3R–amylin and related CTR/RAMP complexes with multiple peptides\",\n      \"pmids\": [\"35324283\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structures are Gs-coupled only; Gq or Gi-coupled states unknown\", \"RAMP3 C-terminal PDZ motif still not resolved structurally\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"RAMP3 was shown to promote ACKR2 membrane expression via Rab4 vesicles and enable CCL2 scavenging in hepatocellular carcinoma cells, extending the RAMP3-dependent recycling mechanism to a second atypical chemokine receptor and linking it to tumor immune microenvironment regulation.\",\n      \"evidence\": \"Co-IP, overexpression/knockdown in HCC cell lines, syngeneic tumors, CCL2 ELISA, flow cytometry for myeloid infiltration\",\n      \"pmids\": [\"39437486\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RAMP3 PDZ motif or another domain mediates ACKR2 interaction not determined\", \"Single tumor model\", \"Independent replication needed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of the RAMP3 C-terminal PDZ motif–NSF interaction, the full inventory of RAMP3-interacting GPCRs, and how RAMP3 biases G-protein coupling selectivity (e.g., at GLP-1R) at a structural level.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal or cryo-EM structure of the RAMP3 PDZ motif with NSF or other scaffold\", \"Comprehensive unbiased interactome screen for RAMP3 across all GPCRs not performed\", \"Mechanism of G-protein coupling bias by RAMP3 structurally undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 8, 17, 24]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 10, 13, 20, 23]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [3, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 2, 14, 17]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2, 8, 17]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [8, 17, 24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 7, 8, 10, 14, 20, 23]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 6, 8]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 8, 17, 24]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [15, 17]}\n    ],\n    \"complexes\": [\n      \"AM2R (CLR/RAMP3)\",\n      \"AMY3R (CTR/RAMP3)\",\n      \"ACKR3/RAMP3\",\n      \"GPR30/RAMP3\"\n    ],\n    \"partners\": [\n      \"CALCRL\",\n      \"CALCR\",\n      \"ACKR3\",\n      \"ACKR2\",\n      \"GPER1\",\n      \"NSF\",\n      \"PTH2R\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}