{"gene":"CALCRL","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":1999,"finding":"The RAMP2/CRLR complex functions as a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells, mediating cAMP elevation upon adrenomedullin stimulation when both RAMP2 and CRLR are co-expressed.","method":"Heterologous co-expression in HeLa EBNA and 293 EBNA cells; cAMP assay; RT-PCR of endogenous expression","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 2 / Strong — functional cAMP assay in co-transfected cells replicated across multiple cell lines; confirmed endogenous co-expression in primary vascular cells","pmids":["10217420"],"is_preprint":false},{"year":2000,"finding":"Rat CRLR co-transfected with RAMP1 cDNA specifies a CGRP receptor, while co-transfection with RAMP2 or RAMP3 specifies an adrenomedullin receptor in HEK293 cells; CGRP binding in rat tissues correlates with RAMP1 mRNA levels and total binding correlates with CRLR mRNA levels.","method":"Co-transfection in HEK293 cells; radioligand binding; quantitative mRNA correlation across eight rat tissues","journal":"British journal of pharmacology","confidence":"High","confidence_rationale":"Tier 2 / Strong — radioligand binding plus functional co-transfection in multiple tissue contexts, replicated findings from other labs","pmids":["10781016"],"is_preprint":false},{"year":2002,"finding":"CRLR does not require heterodimerization with RAMPs for efficient cell surface expression in Xenopus oocytes; RAMP2 and RAMP3 require N-glycosylation for plasma membrane targeting; RAMP1 (not N-glycosylated) reaches the cell surface only upon heterodimer formation with CRLR; introduction of N-glycosylation sites into RAMP1 allows RAMP1 cell surface expression independent of CRLR. These data argue against a chaperone function for RAMPs.","method":"Xenopus oocyte expression system; quantitative cell surface binding assay; site-directed mutagenesis of N-glycosylation sites in RAMP1","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution in oocyte system with mutagenesis, multiple orthogonal approaches (binding assay + glycosylation mutants) in one rigorous study","pmids":["11854283"],"is_preprint":false},{"year":2001,"finding":"Glycosylation of CRLR at Asn123 is required for ligand binding and signal transduction; mutation of Asn123 (but not Asn66 or Asn118) abolishes adrenomedullin/CGRP binding and signaling and reduces cell surface expression; blocking all N-glycosylation with tunicamycin abolishes ligand binding regardless of which RAMP is co-expressed.","method":"Site-directed mutagenesis of CRLR N-glycosylation sites (N66Q, N118Q, N123Q); radioligand binding in HEK293 EBNA and CHO-K1 cells; cAMP signaling assay; FACS cell surface expression analysis","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 / Strong — active-site mutagenesis plus functional assays plus surface expression in multiple cell lines in one study","pmids":["11389975"],"is_preprint":false},{"year":2001,"finding":"CRLR and RAMP2 co-expression is required for functional adrenomedullin (AM) signaling in rat cardiomyocytes; overexpression of either CRLR or RAMP2 alone potentiates AM-stimulated CRE-luciferase activity, with co-overexpression producing an additive ~4-fold increase; AM signaling is abolished by the antagonist CGRP(8-37) or by CRLR antisense.","method":"Transient transfection with CRE-luciferase reporter in neonatal rat cardiomyocytes; CRLR antisense; receptor antagonist treatment; RT-PCR","journal":"Peptides","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter gene functional assay plus antisense knockdown plus pharmacological antagonism, single lab","pmids":["11754972"],"is_preprint":false},{"year":2003,"finding":"The human CRLR gene promoter contains an HIF-1alpha-responsive hypoxia response element (HRE); mutation of the HRE abolishes hypoxia-induced promoter activation; hypoxia transcriptionally upregulates CRLR mRNA in microvascular endothelial cells, while RAMP1, RAMP2, and RAMP3 expression is unaffected by hypoxia.","method":"5'-RACE; promoter cloning; luciferase reporter assay in primary microvascular endothelial cells; HRE site-directed mutagenesis; semi-quantitative RT-PCR","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — promoter cloning with mutagenesis of defined regulatory element plus reporter assay plus mRNA measurement in primary cells","pmids":["12824306"],"is_preprint":false},{"year":2003,"finding":"N-glycosylation of RAMP3 (at two to four of four consensus sites) and disulfide bonds formed by all six conserved cysteine residues are required for functional CRLR/RAMP3 adrenomedullin receptor expression; elimination of all N-glycans significantly reduces [125I]AM binding and increases EC50; mutation of all six cysteines abolishes adrenomedullin binding completely.","method":"Xenopus oocyte expression system; mutagenesis of N-glycosylation sites and cysteine residues in RAMP3; [125I]AM radioligand binding; EC50 determination","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution in oocytes with systematic mutagenesis and radioligand binding in one rigorous study","pmids":["12939163"],"is_preprint":false},{"year":2005,"finding":"CGRP exerts antiapoptotic effects on H9c2 cardiomyoblasts under oxidative stress specifically via the RAMP1/CRLR complex: CGRP prevents caspase-3 activation, phosphatidylserine externalization, DNA fragmentation, and Bax upregulation, increases Bcl-2 expression; these effects are blocked by the RAMP1/RAMP2 inhibitor CGRP(8-37) but not mimicked by adrenomedullin (a RAMP2 agonist), indicating RAMP1/CRLR specificity.","method":"MTT cell viability; annexin V/propidium iodide flow cytometry; caspase-3 activity assay; DNA fragmentation; RT-PCR for Bcl-2/Bax; dot blotting; pharmacological antagonism with CGRP(8-37)","journal":"Journal of molecular and cellular cardiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal functional assays with pharmacological specificity testing, single lab","pmids":["16242145"],"is_preprint":false},{"year":2007,"finding":"CRLR and RAMP1 selectively associate as heterodimers at the cell surface in living cells; CRLR and RAMP1 can also independently form homodimers; CRLR engages G proteins and β-arrestin following CGRP stimulation only in the presence of RAMP1; a RAMP1 mutant unable to reach the cell surface still heterodimerizes with CRLR, indicating that deficient targeting results from altered complex conformation rather than impaired heterodimerization.","method":"BRET (bioluminescence resonance energy transfer) titration in living cells; radioligand binding; cAMP production assay; BRET with G protein and β-arrestin biosensors","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — BRET quantitative protein-protein interaction in living cells combined with functional signaling assays and mutant analysis in one rigorous study","pmids":["17503773"],"is_preprint":false},{"year":2004,"finding":"AM promotes HUVEC migration, invasion, and differentiation into cord-like structures via CRLR/RAMP2 and CRLR/RAMP3 receptors; suboptimal concentrations of VEGF and AM act synergistically; AM-induced capillary tube formation is not blocked by anti-VEGF antibodies, indicating AM does not act indirectly through VEGF upregulation.","method":"Migration and invasion assays; Matrigel cord formation assay; blocking antibody experiments; cAMP assay in HUVEC","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional angiogenesis assays with receptor-specific blocking, single lab","pmids":["14712479"],"is_preprint":false},{"year":2016,"finding":"The hypotensive response to AM is primarily mediated through the CLR/RAMP1 heterodimer in vivo; CGRP's hypotensive activity is predominantly through CLR/RAMP1; CLR/RAMP2 and CLR/RAMP3 also contribute to AM-mediated hypotension; Calcrl+/- mice show attenuated hypotensive response to AM and sex-dependent differences in basal blood pressure.","method":"Genetic mouse models (Ramp1-/-, Ramp2+/-, Ramp3-/-, double KO, Calcrl+/-); intravenous injection of AM or CGRP; blood pressure measurement under anesthesia","journal":"Peptides","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic epistasis with multiple knockout lines and direct physiological readout","pmids":["27940069"],"is_preprint":false},{"year":2020,"finding":"Crystal structure of erenumab (anti-CGRPR monoclonal antibody) in complex with CGRPR reveals that erenumab's 21-residue CDR-H3 loop projects into the deep interface between the CLR and RAMP1 subunits of CGRPR, directly blocking ligand binding; erenumab engages residues specific to both CLR and RAMP1, providing the molecular basis for receptor selectivity.","method":"X-ray crystallography of erenumab–CGRPR complex; functional binding validation","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with atomic-level detail of the CLR–RAMP1 interface, validated functionally","pmids":["32049005"],"is_preprint":false},{"year":2019,"finding":"CGRP acting through CALCRL/RAMP1 protects AML cells from apoptosis induced by cytostatic drugs; specific CGRP antagonists block this protective effect; CGRP/CALCRL promotes leukemic stem cell properties; the CGRP antagonist olcegepant increases differentiation and reduces leukemic burden in a mouse model of AML.","method":"CALCRL expression analysis in AML cell lines and primary samples; pharmacological inhibition with olcegepant; C57BL/6 AML mouse model; apoptosis assays; flow cytometry for stem cell markers","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition plus in vivo mouse model, multiple functional readouts, single lab","pmids":["31756985"],"is_preprint":false},{"year":2021,"finding":"The ADM-CALCRL axis drives cell cycle progression, DNA repair, and mitochondrial OxPHOS function in AML blasts dependent on E2F1 and BCL2 signaling; CALCRL knockdown impairs leukemic growth, decreases LSC frequency, and sensitizes to cytarabine in patient-derived xenograft models; CALCRL depletion reduces LSC frequency of relapse-initiating cells post-chemotherapy in vivo.","method":"shRNA/siRNA CALCRL knockdown; patient-derived xenograft (PDX) models; in vivo cytarabine treatment; transcriptomic analysis identifying E2F1 and BCL2 dependence; cell cycle and mitochondrial function assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo PDX models plus multiple orthogonal mechanistic assays (cell cycle, DNA repair, OxPHOS, E2F1/BCL2 pathway) in one rigorous study","pmids":["33462236"],"is_preprint":false},{"year":2019,"finding":"CRISPR-Cas9-mediated knockout of CALCRL significantly impairs colony formation in human myeloid leukemia cell lines; CALCRL levels positively correlate with leukemic engraftment capacity of primary patient samples in immunocompromised mice.","method":"CRISPR-Cas9 knockout; colony formation assay; patient-derived xenograft engraftment assay","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with defined cellular phenotype plus in vivo engraftment, single lab","pmids":["31182782"],"is_preprint":false},{"year":2019,"finding":"CGRP-CALCRL/RAMP1 signaling is important for maintaining hematopoiesis under proliferative stress: Ramp1-/- mice show decreased bone marrow repopulation capacity and low proliferation with enhanced ROS production and apoptosis following transplantation stress; steady-state hematopoiesis is largely maintained in Ramp1-/- mice.","method":"Ramp1-/- mouse model; bone marrow transplantation assay; cell proliferation and apoptosis assays; ROS measurement","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO mouse model with in vivo hematopoietic functional readout (transplantation), single lab","pmids":["30674976"],"is_preprint":false},{"year":2019,"finding":"Mutant RAMP2 proteins aggregate in transfected cells and disrupt the AM-RAMP2/CRLR-cAMP signaling pathway; ablation of one Ramp2 allele in mice leads to cAMP reduction and retinal ganglion cell death, establishing that the RAMP2/CRLR-cAMP axis is required for retinal ganglion cell survival.","method":"Exome sequencing; functional transfection studies with RAMP2 mutants; heterozygous Ramp2 knockout mouse model; cAMP measurement; retinal ganglion cell survival assay","journal":"Genetics in medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic mouse model plus functional cell-based assay with mutant proteins, single lab","pmids":["31000793"],"is_preprint":false},{"year":2005,"finding":"Acute hypoxia in coronary artery smooth muscle cells (CASMC) rapidly increases CRLR protein level (~3.5-fold within 1 hour) independently of changes in CRLR mRNA, indicating a major post-transcriptional regulatory mechanism; later mRNA elevation was also observed; chronic hypoxia in rat ventricles upregulates both mRNA and protein levels of CRLR and all three RAMPs.","method":"RT-PCR for mRNA; Western blotting for protein; in vitro CASMC hypoxia model; in vivo chronic hypobaric hypoxia rat model","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mRNA and protein dissociation demonstrated by parallel RT-PCR and Western blot in two experimental models, single lab","pmids":["15567147"],"is_preprint":false},{"year":2024,"finding":"CALCRL mediates eNOS, APLN (apelin), angiopoietin, prostaglandin, and EDN1 (endothelin-1) signaling pathways in endothelial cells; HSF1 binds a shear stress-responsive enhancer harboring rs880890 to regulate CALCRL transcription; CRISPR deletion of this enhancer downregulates CALCRL expression; CALCRL knockdown decreases endothelial cell proliferation, tube formation, and NO production.","method":"CRISPR enhancer deletion; HSF1 siRNA knockdown; chromatin immunoprecipitation (ChIP-qPCR); ATAC-seq; luciferase reporter assay; electromobility shift assay (EMSA); CALCRL siRNA knockdown with functional readouts (proliferation, tube formation, NO production)","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal epigenomic and functional methods (CRISPR, ChIP, EMSA, reporter assay, functional cell assays) in one rigorous study","pmids":["38602103"],"is_preprint":false},{"year":2023,"finding":"CALCRL overexpression in AML cells confers resistance to daunorubicin, reduces DNA damage and G0/G1 cell cycle arrest, and reduces apoptosis; mechanistically, CALCRL overexpression upregulates XRCC5 and PDK1 expression and increases AKT/PKCε phosphorylation; XRCC5 siRNA knockdown in CALCRL-overexpressing cells restores drug sensitivity and reverses downstream AKT/PKCε phosphorylation.","method":"CALCRL overexpression constructs in HL-60 and Molm-13 cells; XRCC5 siRNA; RT-PCR; Western blot; cell viability, apoptosis, cell cycle assays; in vivo xenograft mouse model","journal":"Anti-cancer drugs","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function plus siRNA rescue with multiple mechanistic readouts, single lab","pmids":["37948318"],"is_preprint":false},{"year":2025,"finding":"Spinal Calcrl+ neurons function as projection neurons that amplify mechanical itch signaling; in chronic itch models, these neurons show enhanced intrinsic excitability, increased Aβ-fiber-evoked excitatory synaptic input, and reduced inhibitory input; chemogenetic activation of Calcrl+ neurons induces mechanical itch sensitization in naïve mice, while chemogenetic inhibition alleviates it in chronic itch models.","method":"Chemogenetic manipulation (DREADD); behavioral itch assays; electrophysiology (synaptic recordings); morphological assays; chronic itch mouse models (allergic contact dermatitis, atopic dermatitis, psoriasis)","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chemogenetic gain- and loss-of-function with electrophysiological and behavioral readouts, single lab","pmids":["41248150"],"is_preprint":false},{"year":2024,"finding":"CGRP acting via CALCRL/CGRPR protects alveolar cells from hyperoxia-induced injury by inducing Ca2+ entry through TRPV1 channels; CGRP enhances non-selective membrane currents through TRPV1; CGRP-induced Ca2+ increase is reduced by inhibiting the PLC/PKC pathway; selective inhibitors of CGRPR or TRPV1 attenuate CGRP-mediated cell proliferation and anti-apoptotic effects.","method":"Digital calcium imaging; patch clamp electrophysiology; TRPV1 siRNA knockdown; pharmacological inhibition of CGRPR, TRPV1, PLC, and PKC; cell viability and apoptosis assays in A549 cells under hyperoxia","journal":"Molecular medicine reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — electrophysiology plus siRNA knockdown plus pharmacological inhibition with multiple orthogonal readouts, single lab","pmids":["38695251"],"is_preprint":false}],"current_model":"CALCRL (CRLR) is a class B GPCR that functions exclusively as an obligate heterodimer with one of three receptor activity-modifying proteins (RAMPs): CRLR/RAMP1 constitutes a CGRP receptor that signals via cAMP, G proteins, and β-arrestin; CRLR/RAMP2 and CRLR/RAMP3 form adrenomedullin receptors; ligand binding and signaling require N-glycosylation of CRLR at Asn123; RAMP1 requires heterodimerization with CRLR for cell surface delivery (due to lack of N-glycosylation), while RAMP2 and RAMP3 can traffic independently via N-glycans; the CRLR promoter is transcriptionally regulated by HIF-1α under hypoxia; and CALCRL drives leukemic stem cell survival, drug resistance (via E2F1/BCL2/OxPHOS and XRCC5/PDK1/AKT axes), vascular responses (eNOS, NO production, angiogenesis), and nociceptive/itch signaling in spinal neurons."},"narrative":{"mechanistic_narrative":"CALCRL (CRLR/CLR) is a class B GPCR that signals only as an obligate heterodimer with a receptor activity-modifying protein (RAMP), and the identity of the bound RAMP determines ligand selectivity: CRLR/RAMP1 constitutes a CGRP receptor, whereas CRLR/RAMP2 and CRLR/RAMP3 form adrenomedullin receptors that elevate cAMP [PMID:10217420, PMID:10781016]. The CRLR/RAMP1 complex couples to G proteins and β-arrestin upon CGRP stimulation, and BRET in living cells shows that CRLR and RAMP1 selectively associate as cell-surface heterodimers; a RAMP1 trafficking-defective mutant still heterodimerizes with CRLR, establishing that surface delivery depends on complex conformation rather than on heterodimerization per se [PMID:17503773]. Receptor maturation and ligand engagement require post-translational modification: N-glycosylation of CRLR at Asn123 is essential for ligand binding and signaling [PMID:11389975], and the RAMP partner contributes its own glycosylation- and disulfide-dependent folding, with RAMP1 reaching the surface only through CRLR pairing because it lacks the N-glycans that allow RAMP2/RAMP3 to traffic independently [PMID:11854283, PMID:12939163]. Through this system CALCRL transduces vascular and cytoprotective signaling: it mediates adrenomedullin-driven endothelial migration, invasion and tube formation cooperatively with VEGF [PMID:14712479], drives eNOS-dependent NO production, proliferation and angiogenesis under transcriptional control of a shear-stress HSF1 enhancer [PMID:38602103], and the CLR/RAMP1 heterodimer mediates the hypotensive responses to both CGRP and adrenomedullin in vivo [PMID:27940069]. CRLR expression is induced by hypoxia, both transcriptionally via an HIF-1α-responsive element in its promoter [PMID:12824306] and post-transcriptionally with rapid protein accumulation [PMID:15567147]. In disease, the ADM/CGRP–CALCRL axis sustains acute myeloid leukemia: CALCRL is required for leukemic colony formation and engraftment [PMID:31182782], drives cell-cycle progression, DNA repair and mitochondrial OxPHOS through E2F1/BCL2 to maintain leukemic stem cells and chemoresistance [PMID:33462236], and confers daunorubicin resistance via an XRCC5/PDK1/AKT axis [PMID:37948318]. CALCRL signaling is also cytoprotective in cardiomyoblasts and alveolar cells and defines spinal projection neurons that amplify mechanical itch [PMID:16242145, PMID:38695251, PMID:41248150].","teleology":[{"year":1999,"claim":"Established that CRLR is not an orphan receptor but acquires defined ligand pharmacology only when paired with a RAMP, identifying the RAMP2/CRLR complex as a functional adrenomedullin receptor.","evidence":"Heterologous co-expression with cAMP assays in HeLa and 293 cells plus endogenous expression in vascular cells","pmids":["10217420"],"confidence":"High","gaps":["Did not resolve the structural basis of RAMP-conferred selectivity","Other RAMP/CRLR combinations not tested in this study"]},{"year":2000,"claim":"Defined the combinatorial logic that the bound RAMP dictates whether CRLR is a CGRP receptor (RAMP1) or an adrenomedullin receptor (RAMP2/3), linking receptor phenotype to tissue RAMP expression.","evidence":"Co-transfection radioligand binding in HEK293 and quantitative mRNA correlation across rat tissues","pmids":["10781016"],"confidence":"High","gaps":["Functional consequences in native tissues inferred from correlation, not perturbation"]},{"year":2001,"claim":"Identified Asn123 N-glycosylation of CRLR as a requirement for ligand binding and signaling, defining a post-translational determinant of receptor competence independent of RAMP identity.","evidence":"Site-directed mutagenesis (N66Q/N118Q/N123Q), radioligand binding, cAMP and FACS surface assays in HEK293 and CHO cells","pmids":["11389975"],"confidence":"High","gaps":["Mechanism by which Asn123 glycan supports binding vs folding not separated","Glycan structure not characterized"]},{"year":2001,"claim":"Demonstrated in a physiologically relevant cardiomyocyte context that AM responsiveness requires CRLR/RAMP2 co-expression, validating the heterodimer model in native cells.","evidence":"CRE-luciferase reporter, CRLR antisense, and CGRP(8-37) antagonism in neonatal rat cardiomyocytes","pmids":["11754972"],"confidence":"Medium","gaps":["Reporter and antisense readouts only; no direct surface complex measurement","Single lab"]},{"year":2002,"claim":"Reframed RAMP function away from a simple chaperone model, showing RAMP1 surface delivery depends on heterodimerization with CRLR because it lacks the N-glycans that allow RAMP2/3 to traffic independently.","evidence":"Xenopus oocyte reconstitution with quantitative surface binding and engineered RAMP1 glycosylation mutants","pmids":["11854283"],"confidence":"High","gaps":["Trafficking machinery interpreting glycan signals not identified"]},{"year":2003,"claim":"Extended the maturation requirements to the RAMP subunit, showing RAMP3 glycosylation and conserved disulfide bonds are needed for a functional CRLR/RAMP3 adrenomedullin receptor.","evidence":"Oocyte expression with systematic RAMP3 glycosylation/cysteine mutagenesis and [125I]AM binding","pmids":["12939163"],"confidence":"High","gaps":["Disulfide connectivity not directly mapped","Generalization to RAMP1/RAMP2 not tested here"]},{"year":2003,"claim":"Connected CRLR expression to oxygen sensing by identifying a functional HIF-1α hypoxia response element in the human promoter, explaining selective CRLR (not RAMP) induction under hypoxia.","evidence":"Promoter cloning, HRE mutagenesis, luciferase reporter and RT-PCR in primary microvascular endothelial cells","pmids":["12824306"],"confidence":"High","gaps":["Downstream physiological consequence of CRLR induction not measured in this study"]},{"year":2004,"claim":"Established a direct angiogenic role for CALCRL receptors, showing AM drives endothelial migration, invasion and tube formation via CRLR/RAMP2 and CRLR/RAMP3 and cooperates with VEGF.","evidence":"Migration, invasion, Matrigel cord and blocking-antibody assays in HUVEC","pmids":["14712479"],"confidence":"Medium","gaps":["Intracellular effectors of the angiogenic response not defined","Single lab"]},{"year":2005,"claim":"Showed RAMP1/CRLR signaling is cytoprotective, with CGRP suppressing oxidative-stress apoptosis through Bcl-2/Bax modulation in a RAMP1-specific manner.","evidence":"Viability, annexin V, caspase-3, DNA fragmentation and RT-PCR with CGRP(8-37) antagonism in H9c2 cells","pmids":["16242145"],"confidence":"Medium","gaps":["Signaling pathway linking receptor to Bcl-2/Bax not delineated","Single lab"]},{"year":2005,"claim":"Revealed a post-transcriptional layer of hypoxic CRLR regulation, with acute hypoxia raising CRLR protein before mRNA, complementing the HIF-1α transcriptional mechanism.","evidence":"Parallel RT-PCR and Western blot in CASMC and chronic hypoxia rat ventricle","pmids":["15567147"],"confidence":"Medium","gaps":["Post-transcriptional mechanism (translation vs stability) not identified","Single lab"]},{"year":2007,"claim":"Provided direct biophysical evidence for selective cell-surface heterodimerization and showed signaling output (G protein and β-arrestin engagement) requires RAMP1, separating heterodimer formation from trafficking competence.","evidence":"BRET titration, radioligand binding, cAMP and biosensor assays in living cells with a trafficking-defective RAMP1 mutant","pmids":["17503773"],"confidence":"High","gaps":["Conformational change driving trafficking not structurally defined","Homodimer functional relevance unclear"]},{"year":2016,"claim":"Assigned in vivo physiological responsibility, showing the CLR/RAMP1 heterodimer is the principal mediator of AM- and CGRP-induced hypotension with RAMP2/3 contributions.","evidence":"Multiple Ramp and Calcrl knockout mouse lines with intravenous peptide challenge and blood pressure measurement","pmids":["27940069"],"confidence":"High","gaps":["Mechanistic basis of sex-dependent basal blood pressure differences unexplained"]},{"year":2019,"claim":"Identified CALCRL as a driver of acute myeloid leukemia, required for colony formation and correlated with leukemic engraftment capacity.","evidence":"CRISPR-Cas9 knockout, colony assays and patient-derived xenograft engraftment","pmids":["31182782"],"confidence":"Medium","gaps":["Downstream pathway not yet defined in this study","Single lab"]},{"year":2019,"claim":"Demonstrated that CGRP–CALCRL/RAMP1 signaling protects AML cells from cytostatic apoptosis and sustains leukemic stem cell properties, nominating it as a therapeutic target.","evidence":"CALCRL expression analysis, olcegepant antagonism and an AML mouse model with stem-cell and apoptosis readouts","pmids":["31756985"],"confidence":"Medium","gaps":["Transcriptional/metabolic effectors not yet mapped here","Single lab"]},{"year":2019,"claim":"Showed the same CGRP-CALCRL/RAMP1 axis maintains normal stress hematopoiesis, distinguishing stress-dependent from steady-state requirements.","evidence":"Ramp1-/- mice with bone marrow transplantation, proliferation, apoptosis and ROS assays","pmids":["30674976"],"confidence":"Medium","gaps":["Whether the protective program overlaps with the leukemic program untested","Single lab"]},{"year":2019,"claim":"Linked the RAMP2/CRLR-cAMP axis to neuronal survival, with RAMP2 mutants disrupting signaling and Ramp2 haploinsufficiency causing retinal ganglion cell death.","evidence":"Exome sequencing, RAMP2 mutant transfection with cAMP assays and a heterozygous Ramp2 knockout mouse","pmids":["31000793"],"confidence":"Medium","gaps":["Causal mutation-to-phenotype chain in patients not fully established","Single lab"]},{"year":2020,"claim":"Provided an atomic-resolution view of the CLR–RAMP1 interface, showing the therapeutic antibody erenumab wedges into the subunit interface to block ligand binding and explain receptor selectivity.","evidence":"X-ray crystallography of the erenumab–CGRPR complex with functional validation","pmids":["32049005"],"confidence":"High","gaps":["Full agonist-bound active-state structure not resolved here"]},{"year":2021,"claim":"Defined the mechanistic program by which ADM-CALCRL sustains leukemic stem cells, coupling the receptor to E2F1/BCL2-dependent cell cycle, DNA repair and OxPHOS and to chemoresistance.","evidence":"shRNA/siRNA knockdown, PDX models with in vivo cytarabine, transcriptomics and metabolic assays","pmids":["33462236"],"confidence":"High","gaps":["Direct receptor-to-E2F1 signaling steps not fully reconstructed"]},{"year":2023,"claim":"Identified an additional drug-resistance effector arm, with CALCRL upregulating XRCC5/PDK1 and AKT/PKCε phosphorylation to confer daunorubicin resistance, reversible by XRCC5 knockdown.","evidence":"CALCRL overexpression and XRCC5 siRNA rescue with viability, apoptosis, cell-cycle assays and xenografts in HL-60/Molm-13 cells","pmids":["37948318"],"confidence":"Medium","gaps":["How receptor signaling induces XRCC5 not defined","Single lab"]},{"year":2024,"claim":"Mapped the endothelial transcriptional control and functional output of CALCRL, showing an HSF1-bound shear-stress enhancer drives expression that supports eNOS/NO, proliferation and angiogenesis.","evidence":"CRISPR enhancer deletion, HSF1 ChIP/EMSA, ATAC-seq, reporter assays and CALCRL knockdown functional readouts","pmids":["38602103"],"confidence":"High","gaps":["Causal contribution of the rs880890 variant in vivo not established"]},{"year":2024,"claim":"Showed CGRP-CALCRL/CGRPR signaling couples to TRPV1-mediated Ca2+ entry via PLC/PKC to protect alveolar cells from hyperoxic injury.","evidence":"Calcium imaging, patch clamp, TRPV1 siRNA and pharmacological inhibition in A549 cells under hyperoxia","pmids":["38695251"],"confidence":"Medium","gaps":["Physical coupling between receptor and TRPV1 not demonstrated","Single lab"]},{"year":2025,"claim":"Defined a neuronal sensory role for Calcrl-expressing spinal projection neurons in amplifying mechanical itch through altered excitability and synaptic balance.","evidence":"Chemogenetic gain/loss-of-function, electrophysiology and behavioral assays in chronic itch mouse models","pmids":["41248150"],"confidence":"Medium","gaps":["Role of CGRP ligand and RAMP partner in these neurons not addressed","Single lab"]},{"year":null,"claim":"How the diverse downstream programs (vascular eNOS/angiogenesis, leukemic E2F1/BCL2 and XRCC5/AKT, neuronal TRPV1 and itch) are selected from a common CRLR/RAMP cAMP-G protein-β-arrestin core remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unified map linking receptor-proximal signaling to cell-type-specific effectors","Active-state agonist-bound receptor structure not available","Whether leukemic and protective hematopoietic programs are mechanistically distinct is untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,1,8]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[1,8]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[8]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,3,8]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,8]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[13,14,19]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[7,12,21]}],"complexes":["CGRP receptor (CRLR/RAMP1)","Adrenomedullin receptor (CRLR/RAMP2)","Adrenomedullin receptor (CRLR/RAMP3)"],"partners":["RAMP1","RAMP2","RAMP3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q16602","full_name":"Calcitonin gene-related peptide type 1 receptor","aliases":["Calcitonin receptor-like receptor","CRLR"],"length_aa":461,"mass_kda":53.0,"function":"G protein-coupled receptor which specificity is determined by its interaction with receptor-activity-modifying proteins (RAMPs) (PubMed:32296767, PubMed:33602864, PubMed:8626685). Together with RAMP1, form the receptor complex for calcitonin-gene-related peptides CALCA/CGRP1 and CALCB/CGRP2 (PubMed:33602864). Together with RAMP2 or RAMP3, function as receptor complexes for adrenomedullin (ADM and ADM2) (PubMed:32296767, PubMed:9620797). Ligand binding causes a conformation change that triggers signaling via guanine nucleotide-binding proteins (G proteins) and modulates the activity of downstream effectors. 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LMPHM1","url":"https://www.omim.org/entry/153100"},{"mim_id":"114190","title":"CALCITONIN RECEPTOR-LIKE RECEPTOR; CALCRL","url":"https://www.omim.org/entry/114190"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"adipose tissue","ntpm":66.3},{"tissue":"lung","ntpm":72.4}],"url":"https://www.proteinatlas.org/search/CALCRL"},"hgnc":{"alias_symbol":["CGRPR","CRLR"],"prev_symbol":[]},"alphafold":{"accession":"Q16602","domains":[{"cath_id":"4.10.1240.10","chopping":"35-128","consensus_level":"high","plddt":88.0274,"start":35,"end":128},{"cath_id":"1.20.1070.10","chopping":"132-396","consensus_level":"high","plddt":86.0955,"start":132,"end":396}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q16602","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q16602-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q16602-F1-predicted_aligned_error_v6.png","plddt_mean":78.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CALCRL","jax_strain_url":"https://www.jax.org/strain/search?query=CALCRL"},"sequence":{"accession":"Q16602","fasta_url":"https://rest.uniprot.org/uniprotkb/Q16602.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q16602/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q16602"}},"corpus_meta":[{"pmid":"10217420","id":"PMC_10217420","title":"The 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These data argue against a chaperone function for RAMPs.\",\n      \"method\": \"Xenopus oocyte expression system; quantitative cell surface binding assay; site-directed mutagenesis of N-glycosylation sites in RAMP1\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution in oocyte system with mutagenesis, multiple orthogonal approaches (binding assay + glycosylation mutants) in one rigorous study\",\n      \"pmids\": [\"11854283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Glycosylation of CRLR at Asn123 is required for ligand binding and signal transduction; mutation of Asn123 (but not Asn66 or Asn118) abolishes adrenomedullin/CGRP binding and signaling and reduces cell surface expression; blocking all N-glycosylation with tunicamycin abolishes ligand binding regardless of which RAMP is co-expressed.\",\n      \"method\": \"Site-directed mutagenesis of CRLR N-glycosylation sites (N66Q, N118Q, N123Q); radioligand binding in HEK293 EBNA and CHO-K1 cells; cAMP signaling assay; FACS cell surface expression analysis\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — active-site mutagenesis plus functional assays plus surface expression in multiple cell lines in one study\",\n      \"pmids\": [\"11389975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CRLR and RAMP2 co-expression is required for functional adrenomedullin (AM) signaling in rat cardiomyocytes; overexpression of either CRLR or RAMP2 alone potentiates AM-stimulated CRE-luciferase activity, with co-overexpression producing an additive ~4-fold increase; AM signaling is abolished by the antagonist CGRP(8-37) or by CRLR antisense.\",\n      \"method\": \"Transient transfection with CRE-luciferase reporter in neonatal rat cardiomyocytes; CRLR antisense; receptor antagonist treatment; RT-PCR\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter gene functional assay plus antisense knockdown plus pharmacological antagonism, single lab\",\n      \"pmids\": [\"11754972\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The human CRLR gene promoter contains an HIF-1alpha-responsive hypoxia response element (HRE); mutation of the HRE abolishes hypoxia-induced promoter activation; hypoxia transcriptionally upregulates CRLR mRNA in microvascular endothelial cells, while RAMP1, RAMP2, and RAMP3 expression is unaffected by hypoxia.\",\n      \"method\": \"5'-RACE; promoter cloning; luciferase reporter assay in primary microvascular endothelial cells; HRE site-directed mutagenesis; semi-quantitative RT-PCR\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — promoter cloning with mutagenesis of defined regulatory element plus reporter assay plus mRNA measurement in primary cells\",\n      \"pmids\": [\"12824306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"N-glycosylation of RAMP3 (at two to four of four consensus sites) and disulfide bonds formed by all six conserved cysteine residues are required for functional CRLR/RAMP3 adrenomedullin receptor expression; elimination of all N-glycans significantly reduces [125I]AM binding and increases EC50; mutation of all six cysteines abolishes adrenomedullin binding completely.\",\n      \"method\": \"Xenopus oocyte expression system; mutagenesis of N-glycosylation sites and cysteine residues in RAMP3; [125I]AM radioligand binding; EC50 determination\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution in oocytes with systematic mutagenesis and radioligand binding in one rigorous study\",\n      \"pmids\": [\"12939163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CGRP exerts antiapoptotic effects on H9c2 cardiomyoblasts under oxidative stress specifically via the RAMP1/CRLR complex: CGRP prevents caspase-3 activation, phosphatidylserine externalization, DNA fragmentation, and Bax upregulation, increases Bcl-2 expression; these effects are blocked by the RAMP1/RAMP2 inhibitor CGRP(8-37) but not mimicked by adrenomedullin (a RAMP2 agonist), indicating RAMP1/CRLR specificity.\",\n      \"method\": \"MTT cell viability; annexin V/propidium iodide flow cytometry; caspase-3 activity assay; DNA fragmentation; RT-PCR for Bcl-2/Bax; dot blotting; pharmacological antagonism with CGRP(8-37)\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal functional assays with pharmacological specificity testing, single lab\",\n      \"pmids\": [\"16242145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CRLR and RAMP1 selectively associate as heterodimers at the cell surface in living cells; CRLR and RAMP1 can also independently form homodimers; CRLR engages G proteins and β-arrestin following CGRP stimulation only in the presence of RAMP1; a RAMP1 mutant unable to reach the cell surface still heterodimerizes with CRLR, indicating that deficient targeting results from altered complex conformation rather than impaired heterodimerization.\",\n      \"method\": \"BRET (bioluminescence resonance energy transfer) titration in living cells; radioligand binding; cAMP production assay; BRET with G protein and β-arrestin biosensors\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — BRET quantitative protein-protein interaction in living cells combined with functional signaling assays and mutant analysis in one rigorous study\",\n      \"pmids\": [\"17503773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"AM promotes HUVEC migration, invasion, and differentiation into cord-like structures via CRLR/RAMP2 and CRLR/RAMP3 receptors; suboptimal concentrations of VEGF and AM act synergistically; AM-induced capillary tube formation is not blocked by anti-VEGF antibodies, indicating AM does not act indirectly through VEGF upregulation.\",\n      \"method\": \"Migration and invasion assays; Matrigel cord formation assay; blocking antibody experiments; cAMP assay in HUVEC\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional angiogenesis assays with receptor-specific blocking, single lab\",\n      \"pmids\": [\"14712479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The hypotensive response to AM is primarily mediated through the CLR/RAMP1 heterodimer in vivo; CGRP's hypotensive activity is predominantly through CLR/RAMP1; CLR/RAMP2 and CLR/RAMP3 also contribute to AM-mediated hypotension; Calcrl+/- mice show attenuated hypotensive response to AM and sex-dependent differences in basal blood pressure.\",\n      \"method\": \"Genetic mouse models (Ramp1-/-, Ramp2+/-, Ramp3-/-, double KO, Calcrl+/-); intravenous injection of AM or CGRP; blood pressure measurement under anesthesia\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic epistasis with multiple knockout lines and direct physiological readout\",\n      \"pmids\": [\"27940069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structure of erenumab (anti-CGRPR monoclonal antibody) in complex with CGRPR reveals that erenumab's 21-residue CDR-H3 loop projects into the deep interface between the CLR and RAMP1 subunits of CGRPR, directly blocking ligand binding; erenumab engages residues specific to both CLR and RAMP1, providing the molecular basis for receptor selectivity.\",\n      \"method\": \"X-ray crystallography of erenumab–CGRPR complex; functional binding validation\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with atomic-level detail of the CLR–RAMP1 interface, validated functionally\",\n      \"pmids\": [\"32049005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CGRP acting through CALCRL/RAMP1 protects AML cells from apoptosis induced by cytostatic drugs; specific CGRP antagonists block this protective effect; CGRP/CALCRL promotes leukemic stem cell properties; the CGRP antagonist olcegepant increases differentiation and reduces leukemic burden in a mouse model of AML.\",\n      \"method\": \"CALCRL expression analysis in AML cell lines and primary samples; pharmacological inhibition with olcegepant; C57BL/6 AML mouse model; apoptosis assays; flow cytometry for stem cell markers\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition plus in vivo mouse model, multiple functional readouts, single lab\",\n      \"pmids\": [\"31756985\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The ADM-CALCRL axis drives cell cycle progression, DNA repair, and mitochondrial OxPHOS function in AML blasts dependent on E2F1 and BCL2 signaling; CALCRL knockdown impairs leukemic growth, decreases LSC frequency, and sensitizes to cytarabine in patient-derived xenograft models; CALCRL depletion reduces LSC frequency of relapse-initiating cells post-chemotherapy in vivo.\",\n      \"method\": \"shRNA/siRNA CALCRL knockdown; patient-derived xenograft (PDX) models; in vivo cytarabine treatment; transcriptomic analysis identifying E2F1 and BCL2 dependence; cell cycle and mitochondrial function assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo PDX models plus multiple orthogonal mechanistic assays (cell cycle, DNA repair, OxPHOS, E2F1/BCL2 pathway) in one rigorous study\",\n      \"pmids\": [\"33462236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CRISPR-Cas9-mediated knockout of CALCRL significantly impairs colony formation in human myeloid leukemia cell lines; CALCRL levels positively correlate with leukemic engraftment capacity of primary patient samples in immunocompromised mice.\",\n      \"method\": \"CRISPR-Cas9 knockout; colony formation assay; patient-derived xenograft engraftment assay\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with defined cellular phenotype plus in vivo engraftment, single lab\",\n      \"pmids\": [\"31182782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CGRP-CALCRL/RAMP1 signaling is important for maintaining hematopoiesis under proliferative stress: Ramp1-/- mice show decreased bone marrow repopulation capacity and low proliferation with enhanced ROS production and apoptosis following transplantation stress; steady-state hematopoiesis is largely maintained in Ramp1-/- mice.\",\n      \"method\": \"Ramp1-/- mouse model; bone marrow transplantation assay; cell proliferation and apoptosis assays; ROS measurement\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO mouse model with in vivo hematopoietic functional readout (transplantation), single lab\",\n      \"pmids\": [\"30674976\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Mutant RAMP2 proteins aggregate in transfected cells and disrupt the AM-RAMP2/CRLR-cAMP signaling pathway; ablation of one Ramp2 allele in mice leads to cAMP reduction and retinal ganglion cell death, establishing that the RAMP2/CRLR-cAMP axis is required for retinal ganglion cell survival.\",\n      \"method\": \"Exome sequencing; functional transfection studies with RAMP2 mutants; heterozygous Ramp2 knockout mouse model; cAMP measurement; retinal ganglion cell survival assay\",\n      \"journal\": \"Genetics in medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic mouse model plus functional cell-based assay with mutant proteins, single lab\",\n      \"pmids\": [\"31000793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Acute hypoxia in coronary artery smooth muscle cells (CASMC) rapidly increases CRLR protein level (~3.5-fold within 1 hour) independently of changes in CRLR mRNA, indicating a major post-transcriptional regulatory mechanism; later mRNA elevation was also observed; chronic hypoxia in rat ventricles upregulates both mRNA and protein levels of CRLR and all three RAMPs.\",\n      \"method\": \"RT-PCR for mRNA; Western blotting for protein; in vitro CASMC hypoxia model; in vivo chronic hypobaric hypoxia rat model\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mRNA and protein dissociation demonstrated by parallel RT-PCR and Western blot in two experimental models, single lab\",\n      \"pmids\": [\"15567147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CALCRL mediates eNOS, APLN (apelin), angiopoietin, prostaglandin, and EDN1 (endothelin-1) signaling pathways in endothelial cells; HSF1 binds a shear stress-responsive enhancer harboring rs880890 to regulate CALCRL transcription; CRISPR deletion of this enhancer downregulates CALCRL expression; CALCRL knockdown decreases endothelial cell proliferation, tube formation, and NO production.\",\n      \"method\": \"CRISPR enhancer deletion; HSF1 siRNA knockdown; chromatin immunoprecipitation (ChIP-qPCR); ATAC-seq; luciferase reporter assay; electromobility shift assay (EMSA); CALCRL siRNA knockdown with functional readouts (proliferation, tube formation, NO production)\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal epigenomic and functional methods (CRISPR, ChIP, EMSA, reporter assay, functional cell assays) in one rigorous study\",\n      \"pmids\": [\"38602103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CALCRL overexpression in AML cells confers resistance to daunorubicin, reduces DNA damage and G0/G1 cell cycle arrest, and reduces apoptosis; mechanistically, CALCRL overexpression upregulates XRCC5 and PDK1 expression and increases AKT/PKCε phosphorylation; XRCC5 siRNA knockdown in CALCRL-overexpressing cells restores drug sensitivity and reverses downstream AKT/PKCε phosphorylation.\",\n      \"method\": \"CALCRL overexpression constructs in HL-60 and Molm-13 cells; XRCC5 siRNA; RT-PCR; Western blot; cell viability, apoptosis, cell cycle assays; in vivo xenograft mouse model\",\n      \"journal\": \"Anti-cancer drugs\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function plus siRNA rescue with multiple mechanistic readouts, single lab\",\n      \"pmids\": [\"37948318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Spinal Calcrl+ neurons function as projection neurons that amplify mechanical itch signaling; in chronic itch models, these neurons show enhanced intrinsic excitability, increased Aβ-fiber-evoked excitatory synaptic input, and reduced inhibitory input; chemogenetic activation of Calcrl+ neurons induces mechanical itch sensitization in naïve mice, while chemogenetic inhibition alleviates it in chronic itch models.\",\n      \"method\": \"Chemogenetic manipulation (DREADD); behavioral itch assays; electrophysiology (synaptic recordings); morphological assays; chronic itch mouse models (allergic contact dermatitis, atopic dermatitis, psoriasis)\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chemogenetic gain- and loss-of-function with electrophysiological and behavioral readouts, single lab\",\n      \"pmids\": [\"41248150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CGRP acting via CALCRL/CGRPR protects alveolar cells from hyperoxia-induced injury by inducing Ca2+ entry through TRPV1 channels; CGRP enhances non-selective membrane currents through TRPV1; CGRP-induced Ca2+ increase is reduced by inhibiting the PLC/PKC pathway; selective inhibitors of CGRPR or TRPV1 attenuate CGRP-mediated cell proliferation and anti-apoptotic effects.\",\n      \"method\": \"Digital calcium imaging; patch clamp electrophysiology; TRPV1 siRNA knockdown; pharmacological inhibition of CGRPR, TRPV1, PLC, and PKC; cell viability and apoptosis assays in A549 cells under hyperoxia\",\n      \"journal\": \"Molecular medicine reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology plus siRNA knockdown plus pharmacological inhibition with multiple orthogonal readouts, single lab\",\n      \"pmids\": [\"38695251\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CALCRL (CRLR) is a class B GPCR that functions exclusively as an obligate heterodimer with one of three receptor activity-modifying proteins (RAMPs): CRLR/RAMP1 constitutes a CGRP receptor that signals via cAMP, G proteins, and β-arrestin; CRLR/RAMP2 and CRLR/RAMP3 form adrenomedullin receptors; ligand binding and signaling require N-glycosylation of CRLR at Asn123; RAMP1 requires heterodimerization with CRLR for cell surface delivery (due to lack of N-glycosylation), while RAMP2 and RAMP3 can traffic independently via N-glycans; the CRLR promoter is transcriptionally regulated by HIF-1α under hypoxia; and CALCRL drives leukemic stem cell survival, drug resistance (via E2F1/BCL2/OxPHOS and XRCC5/PDK1/AKT axes), vascular responses (eNOS, NO production, angiogenesis), and nociceptive/itch signaling in spinal neurons.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CALCRL (CRLR/CLR) is a class B GPCR that signals only as an obligate heterodimer with a receptor activity-modifying protein (RAMP), and the identity of the bound RAMP determines ligand selectivity: CRLR/RAMP1 constitutes a CGRP receptor, whereas CRLR/RAMP2 and CRLR/RAMP3 form adrenomedullin receptors that elevate cAMP [#0, #1]. The CRLR/RAMP1 complex couples to G proteins and \\u03b2-arrestin upon CGRP stimulation, and BRET in living cells shows that CRLR and RAMP1 selectively associate as cell-surface heterodimers; a RAMP1 trafficking-defective mutant still heterodimerizes with CRLR, establishing that surface delivery depends on complex conformation rather than on heterodimerization per se [#8]. Receptor maturation and ligand engagement require post-translational modification: N-glycosylation of CRLR at Asn123 is essential for ligand binding and signaling [#3], and the RAMP partner contributes its own glycosylation- and disulfide-dependent folding, with RAMP1 reaching the surface only through CRLR pairing because it lacks the N-glycans that allow RAMP2/RAMP3 to traffic independently [#2, #6]. Through this system CALCRL transduces vascular and cytoprotective signaling: it mediates adrenomedullin-driven endothelial migration, invasion and tube formation cooperatively with VEGF [#9], drives eNOS-dependent NO production, proliferation and angiogenesis under transcriptional control of a shear-stress HSF1 enhancer [#18], and the CLR/RAMP1 heterodimer mediates the hypotensive responses to both CGRP and adrenomedullin in vivo [#10]. CRLR expression is induced by hypoxia, both transcriptionally via an HIF-1\\u03b1-responsive element in its promoter [#5] and post-transcriptionally with rapid protein accumulation [#17]. In disease, the ADM/CGRP\\u2013CALCRL axis sustains acute myeloid leukemia: CALCRL is required for leukemic colony formation and engraftment [#14], drives cell-cycle progression, DNA repair and mitochondrial OxPHOS through E2F1/BCL2 to maintain leukemic stem cells and chemoresistance [#13], and confers daunorubicin resistance via an XRCC5/PDK1/AKT axis [#19]. CALCRL signaling is also cytoprotective in cardiomyoblasts and alveolar cells and defines spinal projection neurons that amplify mechanical itch [#7, #21, #20].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established that CRLR is not an orphan receptor but acquires defined ligand pharmacology only when paired with a RAMP, identifying the RAMP2/CRLR complex as a functional adrenomedullin receptor.\",\n      \"evidence\": \"Heterologous co-expression with cAMP assays in HeLa and 293 cells plus endogenous expression in vascular cells\",\n      \"pmids\": [\"10217420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of RAMP-conferred selectivity\", \"Other RAMP/CRLR combinations not tested in this study\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defined the combinatorial logic that the bound RAMP dictates whether CRLR is a CGRP receptor (RAMP1) or an adrenomedullin receptor (RAMP2/3), linking receptor phenotype to tissue RAMP expression.\",\n      \"evidence\": \"Co-transfection radioligand binding in HEK293 and quantitative mRNA correlation across rat tissues\",\n      \"pmids\": [\"10781016\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequences in native tissues inferred from correlation, not perturbation\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified Asn123 N-glycosylation of CRLR as a requirement for ligand binding and signaling, defining a post-translational determinant of receptor competence independent of RAMP identity.\",\n      \"evidence\": \"Site-directed mutagenesis (N66Q/N118Q/N123Q), radioligand binding, cAMP and FACS surface assays in HEK293 and CHO cells\",\n      \"pmids\": [\"11389975\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which Asn123 glycan supports binding vs folding not separated\", \"Glycan structure not characterized\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrated in a physiologically relevant cardiomyocyte context that AM responsiveness requires CRLR/RAMP2 co-expression, validating the heterodimer model in native cells.\",\n      \"evidence\": \"CRE-luciferase reporter, CRLR antisense, and CGRP(8-37) antagonism in neonatal rat cardiomyocytes\",\n      \"pmids\": [\"11754972\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reporter and antisense readouts only; no direct surface complex measurement\", \"Single lab\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Reframed RAMP function away from a simple chaperone model, showing RAMP1 surface delivery depends on heterodimerization with CRLR because it lacks the N-glycans that allow RAMP2/3 to traffic independently.\",\n      \"evidence\": \"Xenopus oocyte reconstitution with quantitative surface binding and engineered RAMP1 glycosylation mutants\",\n      \"pmids\": [\"11854283\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trafficking machinery interpreting glycan signals not identified\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Extended the maturation requirements to the RAMP subunit, showing RAMP3 glycosylation and conserved disulfide bonds are needed for a functional CRLR/RAMP3 adrenomedullin receptor.\",\n      \"evidence\": \"Oocyte expression with systematic RAMP3 glycosylation/cysteine mutagenesis and [125I]AM binding\",\n      \"pmids\": [\"12939163\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Disulfide connectivity not directly mapped\", \"Generalization to RAMP1/RAMP2 not tested here\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Connected CRLR expression to oxygen sensing by identifying a functional HIF-1\\u03b1 hypoxia response element in the human promoter, explaining selective CRLR (not RAMP) induction under hypoxia.\",\n      \"evidence\": \"Promoter cloning, HRE mutagenesis, luciferase reporter and RT-PCR in primary microvascular endothelial cells\",\n      \"pmids\": [\"12824306\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream physiological consequence of CRLR induction not measured in this study\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Established a direct angiogenic role for CALCRL receptors, showing AM drives endothelial migration, invasion and tube formation via CRLR/RAMP2 and CRLR/RAMP3 and cooperates with VEGF.\",\n      \"evidence\": \"Migration, invasion, Matrigel cord and blocking-antibody assays in HUVEC\",\n      \"pmids\": [\"14712479\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Intracellular effectors of the angiogenic response not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed RAMP1/CRLR signaling is cytoprotective, with CGRP suppressing oxidative-stress apoptosis through Bcl-2/Bax modulation in a RAMP1-specific manner.\",\n      \"evidence\": \"Viability, annexin V, caspase-3, DNA fragmentation and RT-PCR with CGRP(8-37) antagonism in H9c2 cells\",\n      \"pmids\": [\"16242145\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Signaling pathway linking receptor to Bcl-2/Bax not delineated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Revealed a post-transcriptional layer of hypoxic CRLR regulation, with acute hypoxia raising CRLR protein before mRNA, complementing the HIF-1\\u03b1 transcriptional mechanism.\",\n      \"evidence\": \"Parallel RT-PCR and Western blot in CASMC and chronic hypoxia rat ventricle\",\n      \"pmids\": [\"15567147\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Post-transcriptional mechanism (translation vs stability) not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Provided direct biophysical evidence for selective cell-surface heterodimerization and showed signaling output (G protein and \\u03b2-arrestin engagement) requires RAMP1, separating heterodimer formation from trafficking competence.\",\n      \"evidence\": \"BRET titration, radioligand binding, cAMP and biosensor assays in living cells with a trafficking-defective RAMP1 mutant\",\n      \"pmids\": [\"17503773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational change driving trafficking not structurally defined\", \"Homodimer functional relevance unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Assigned in vivo physiological responsibility, showing the CLR/RAMP1 heterodimer is the principal mediator of AM- and CGRP-induced hypotension with RAMP2/3 contributions.\",\n      \"evidence\": \"Multiple Ramp and Calcrl knockout mouse lines with intravenous peptide challenge and blood pressure measurement\",\n      \"pmids\": [\"27940069\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic basis of sex-dependent basal blood pressure differences unexplained\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified CALCRL as a driver of acute myeloid leukemia, required for colony formation and correlated with leukemic engraftment capacity.\",\n      \"evidence\": \"CRISPR-Cas9 knockout, colony assays and patient-derived xenograft engraftment\",\n      \"pmids\": [\"31182782\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Downstream pathway not yet defined in this study\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated that CGRP\\u2013CALCRL/RAMP1 signaling protects AML cells from cytostatic apoptosis and sustains leukemic stem cell properties, nominating it as a therapeutic target.\",\n      \"evidence\": \"CALCRL expression analysis, olcegepant antagonism and an AML mouse model with stem-cell and apoptosis readouts\",\n      \"pmids\": [\"31756985\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcriptional/metabolic effectors not yet mapped here\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed the same CGRP-CALCRL/RAMP1 axis maintains normal stress hematopoiesis, distinguishing stress-dependent from steady-state requirements.\",\n      \"evidence\": \"Ramp1-/- mice with bone marrow transplantation, proliferation, apoptosis and ROS assays\",\n      \"pmids\": [\"30674976\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the protective program overlaps with the leukemic program untested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linked the RAMP2/CRLR-cAMP axis to neuronal survival, with RAMP2 mutants disrupting signaling and Ramp2 haploinsufficiency causing retinal ganglion cell death.\",\n      \"evidence\": \"Exome sequencing, RAMP2 mutant transfection with cAMP assays and a heterozygous Ramp2 knockout mouse\",\n      \"pmids\": [\"31000793\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal mutation-to-phenotype chain in patients not fully established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided an atomic-resolution view of the CLR\\u2013RAMP1 interface, showing the therapeutic antibody erenumab wedges into the subunit interface to block ligand binding and explain receptor selectivity.\",\n      \"evidence\": \"X-ray crystallography of the erenumab\\u2013CGRPR complex with functional validation\",\n      \"pmids\": [\"32049005\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full agonist-bound active-state structure not resolved here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined the mechanistic program by which ADM-CALCRL sustains leukemic stem cells, coupling the receptor to E2F1/BCL2-dependent cell cycle, DNA repair and OxPHOS and to chemoresistance.\",\n      \"evidence\": \"shRNA/siRNA knockdown, PDX models with in vivo cytarabine, transcriptomics and metabolic assays\",\n      \"pmids\": [\"33462236\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct receptor-to-E2F1 signaling steps not fully reconstructed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified an additional drug-resistance effector arm, with CALCRL upregulating XRCC5/PDK1 and AKT/PKC\\u03b5 phosphorylation to confer daunorubicin resistance, reversible by XRCC5 knockdown.\",\n      \"evidence\": \"CALCRL overexpression and XRCC5 siRNA rescue with viability, apoptosis, cell-cycle assays and xenografts in HL-60/Molm-13 cells\",\n      \"pmids\": [\"37948318\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How receptor signaling induces XRCC5 not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Mapped the endothelial transcriptional control and functional output of CALCRL, showing an HSF1-bound shear-stress enhancer drives expression that supports eNOS/NO, proliferation and angiogenesis.\",\n      \"evidence\": \"CRISPR enhancer deletion, HSF1 ChIP/EMSA, ATAC-seq, reporter assays and CALCRL knockdown functional readouts\",\n      \"pmids\": [\"38602103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Causal contribution of the rs880890 variant in vivo not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed CGRP-CALCRL/CGRPR signaling couples to TRPV1-mediated Ca2+ entry via PLC/PKC to protect alveolar cells from hyperoxic injury.\",\n      \"evidence\": \"Calcium imaging, patch clamp, TRPV1 siRNA and pharmacological inhibition in A549 cells under hyperoxia\",\n      \"pmids\": [\"38695251\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physical coupling between receptor and TRPV1 not demonstrated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a neuronal sensory role for Calcrl-expressing spinal projection neurons in amplifying mechanical itch through altered excitability and synaptic balance.\",\n      \"evidence\": \"Chemogenetic gain/loss-of-function, electrophysiology and behavioral assays in chronic itch mouse models\",\n      \"pmids\": [\"41248150\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Role of CGRP ligand and RAMP partner in these neurons not addressed\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the diverse downstream programs (vascular eNOS/angiogenesis, leukemic E2F1/BCL2 and XRCC5/AKT, neuronal TRPV1 and itch) are selected from a common CRLR/RAMP cAMP-G protein-\\u03b2-arrestin core remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified map linking receptor-proximal signaling to cell-type-specific effectors\", \"Active-state agonist-bound receptor structure not available\", \"Whether leukemic and protective hematopoietic programs are mechanistically distinct is untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 8]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [1, 8]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 3, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 8]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [13, 14, 19]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [7, 12, 21]}\n    ],\n    \"complexes\": [\"CGRP receptor (CRLR/RAMP1)\", \"Adrenomedullin receptor (CRLR/RAMP2)\", \"Adrenomedullin receptor (CRLR/RAMP3)\"],\n    \"partners\": [\"RAMP1\", \"RAMP2\", \"RAMP3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}