{"gene":"CALCRL","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1999,"finding":"The RAMP2/CRLR (CALCRL) complex functions as a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells, mediating cAMP elevation upon adrenomedullin stimulation.","method":"Co-transfection of RAMP2 and CRLR in HeLa EBNA and 293 EBNA cells with cAMP functional assay","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 2 — functional receptor reconstitution in heterologous cells, replicated across multiple labs","pmids":["10217420"],"is_preprint":false},{"year":2000,"finding":"Rat CRLR forms a CGRP receptor when co-expressed with RAMP1, and an adrenomedullin receptor when co-expressed with RAMP2 or RAMP3, establishing RAMP identity as the determinant of ligand specificity for CALCRL.","method":"Co-transfection in HEK293 cells with radioligand binding and cAMP functional assays; correlation of RAMP mRNA with binding in rat tissues","journal":"British journal of pharmacology","confidence":"High","confidence_rationale":"Tier 1/2 — multiple orthogonal methods (binding + functional assay + tissue correlation), independently replicated","pmids":["10781016"],"is_preprint":false},{"year":2002,"finding":"CRLR cell-surface expression does not require heterodimer assembly with RAMPs; RAMP2 and RAMP3 reach the plasma membrane via N-glycosylation-dependent mechanisms, while RAMP1 (non-glycosylated) requires heterodimerization with CRLR for plasma membrane targeting.","method":"Xenopus oocyte expression system with quantitative cell-surface binding assay; N-glycosylation site mutagenesis of RAMP1","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstitution in Xenopus oocytes with mutagenesis and quantitative surface detection","pmids":["11854283"],"is_preprint":false},{"year":2001,"finding":"Glycosylation of CRLR at Asn123 is required for ligand binding and signal transduction; loss of glycosylation at this site impairs cell-surface transport and receptor conformation without affecting expression of N66Q or N118Q mutants.","method":"Site-directed mutagenesis of N-glycosylation sites, radioligand binding, FACS analysis in HEK293 EBNA and CHO-K1 cells","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis combined with binding and surface expression assays","pmids":["11389975"],"is_preprint":false},{"year":2003,"finding":"N-glycosylation and conserved cysteine residues in RAMP3 are critical for functional expression of the CRLR/RAMP3 adrenomedullin receptor; elimination of all N-glycans reduces AM binding, and mutation of all six cysteines abolishes adrenomedullin binding.","method":"Xenopus oocyte expression system with [125I]AM radioligand binding, N-glycosylation site and cysteine mutagenesis","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstitution with systematic mutagenesis and binding assay","pmids":["12939163"],"is_preprint":false},{"year":2007,"finding":"CRLR and RAMP1 form selective heterodimers at the cell surface (not random oligomers); both CRLR and RAMP1 can also form homodimers. CRLR recruits G proteins and β-arrestin upon CGRP stimulation only in the presence of RAMP1.","method":"BRET titration assays in living cells; radioligand binding; cAMP production assays with CRLR-Rluc and RAMP1-GFP fusion proteins","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal BRET methods plus functional assays in living cells","pmids":["17503773"],"is_preprint":false},{"year":2001,"finding":"Adrenomedullin signaling in rat cardiomyocytes requires CRLR and RAMP2 to constitute functional AM receptors; overexpression of CRLR or RAMP2 potentiates AM-induced CRE-luciferase (cAMP) signaling, and CRLR antisense abolishes AM response.","method":"Transient transfection of cardiomyocytes with CRE-luciferase reporter; CRLR antisense and receptor antagonist CGRP(8-37) blockade","journal":"Peptides","confidence":"High","confidence_rationale":"Tier 2 — functional reporter assay with antisense knockdown and pharmacological antagonism","pmids":["11754972"],"is_preprint":false},{"year":2003,"finding":"The human CRLR gene promoter contains a functional hypoxia-response element (HRE) that is activated by HIF-1α under hypoxia; site-directed mutagenesis of the HRE abolishes hypoxia-induced promoter activity in primary microvascular endothelial cells.","method":"5'-RACE, promoter cloning, reporter gene assays, site-directed mutagenesis of HRE, semi-quantitative RT-PCR under hypoxic conditions","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis of HRE combined with reporter assays and mRNA quantification","pmids":["12824306"],"is_preprint":false},{"year":2004,"finding":"CRLR/RAMP2 and CRLR/RAMP3 receptors mediate adrenomedullin-induced HUVEC migration, invasion, and differentiation into cord-like structures during angiogenesis; AM acts independently of VEGF in capillary tube formation.","method":"HUVEC migration and invasion assays, Matrigel differentiation assay, blocking antibodies and receptor-specific pharmacology","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 — multiple cellular assays with pharmacological blockade, single lab","pmids":["14712479"],"is_preprint":false},{"year":2005,"finding":"CGRP exerts antiapoptotic effects in H9c2 cardiomyoblasts specifically through the RAMP1/CRLR complex, as shown by CGRP(8-37) antagonist blockade; RAMP1 expression is upregulated by CGRP and CGRP prevents oxidative stress-induced Bcl-2 decrease and Bax increase.","method":"MTT assays, caspase-3 activation, DNA fragmentation, RT-PCR, dot blot; CGRP(8-37) antagonist and adrenomedullin comparison in H9c2 cells","journal":"Journal of molecular and cellular cardiology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal assays with pharmacological blockade, single lab","pmids":["16242145"],"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 interface between CLR (CALCRL) and RAMP1, directly blocking ligand binding by contacting residues specific to both CLR and RAMP1.","method":"Crystal structure determination of erenumab-CGRPR complex","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional validation of direct ligand-blocking mechanism","pmids":["32049005"],"is_preprint":false},{"year":2016,"finding":"The hypotensive response to adrenomedullin is primarily mediated through CLR/RAMP1, with contributions from CLR/RAMP2 and CLR/RAMP3; CGRP hypotension is predominantly through CLR/RAMP1. Genetic reduction of Calcrl attenuates the hypotensive response to both AM and CGRP in vivo.","method":"Blood pressure measurement in Ramp1-/-, Ramp2+/-, Ramp3-/-, Ramp1-/-/Ramp3-/- double-KO, and Calcrl+/- mice with intravenous AM and CGRP injection","journal":"Peptides","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis in multiple KO/KD mouse models with defined cardiovascular phenotype","pmids":["27940069"],"is_preprint":false},{"year":2019,"finding":"CALCRL knockdown by CRISPR-Cas9 significantly impairs colony formation in human myeloid leukemia cell lines, establishing a functional role for CALCRL in leukemic cell growth.","method":"CRISPR-Cas9 knockout in human AML cell lines with colony formation assay","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with defined cellular phenotype, single lab","pmids":["31182782"],"is_preprint":false},{"year":2019,"finding":"The CGRP-CALCRL/RAMP1 axis protects AML cells from chemotherapy-induced apoptosis; CGRP antagonist olcegepant increases differentiation, reduces leukemic burden, and decreases stem cell properties in a mouse AML model.","method":"Apoptosis assays in AML cell lines and primary samples; CGRP antagonist (olcegepant) treatment; C57BL/6 mouse AML model with in vivo leukemic burden and stem cell assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — multiple functional assays with pharmacological antagonism and in vivo model, 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; CALCRL knockdown decreases leukemic stem cell frequency and sensitizes to cytarabine in patient-derived xenograft models.","method":"CALCRL knockdown in patient-derived xenograft models; cell cycle, DNA repair, and OxPHOS functional assays; in vivo LSC frequency assays post-chemotherapy","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal mechanistic assays plus in vivo patient-derived xenograft models","pmids":["33462236"],"is_preprint":false},{"year":2019,"finding":"CGRP-CALCRL/RAMP1 signaling is required for stress-induced hematopoiesis; Ramp1-deficient mice show decreased bone marrow repopulation capacity, reduced proliferation, enhanced ROS production, and increased apoptosis under proliferative stress.","method":"Ramp1-/- mouse model; BM transplantation/repopulation assay; ROS measurement; apoptosis assay; CGRP administration experiments","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO model with multiple functional readouts, single lab","pmids":["30674976"],"is_preprint":false},{"year":2019,"finding":"Mutant RAMP2 proteins aggregate in transfected cells and disrupt AM-RAMP2/CRLR-cAMP signaling; ablation of one Ramp2 allele leads to cAMP reduction and retinal ganglion cell death in mice, establishing that the RAMP2/CRLR-cAMP axis is essential for retinal ganglion cell survival.","method":"Heterozygous Ramp2 knockout mouse model; transfection of mutant RAMP2 constructs; cAMP measurement; retinal ganglion cell death assay","journal":"Genetics in medicine","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse model combined with functional cAMP assay and cell death quantification","pmids":["31000793"],"is_preprint":false},{"year":2005,"finding":"Acute hypoxia in coronary artery smooth muscle cells induces a rapid increase in CRLR protein independently of changes in CRLR mRNA, indicating a post-transcriptional regulatory mechanism; chronic hypoxia in rats enhances both mRNA and protein of CRLR and all three RAMPs in cardiac ventricles.","method":"Western blotting and RT-PCR in human coronary artery smooth muscle cells under acute hypoxia; chronic hypobaric hypoxia rat model","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — two complementary experimental models with protein and mRNA quantification","pmids":["15567147"],"is_preprint":false},{"year":2024,"finding":"CALCRL expression in endothelial cells is regulated by an HSF1-bound shear stress-responsive enhancer at rs880890; CRISPR deletion of this enhancer downregulates CALCRL expression, and CALCRL knockdown reduces eNOS, apelin, angiopoietin, prostaglandin, and EDN1 signaling, decreasing cell proliferation, tube formation, and NO production.","method":"CRISPR enhancer deletion, siRNA knockdown, ATAC-seq, ChIP-qPCR, electromobility shift assay, luciferase reporter assay, functional proliferation and tube formation assays in human aortic endothelial cells","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"High","confidence_rationale":"Tier 1/2 — multiple orthogonal epigenomic, genetic, and functional methods in a single study","pmids":["38602103"],"is_preprint":false},{"year":2023,"finding":"CALCRL overexpression in AML cells confers resistance to daunorubicin through upregulation of XRCC5 and PDK1, leading to increased AKT/PKCε phosphorylation; XRCC5 siRNA in CALCRL-overexpressing cells restores drug sensitivity and increases apoptosis.","method":"CALCRL overexpression constructs in HL-60 and Molm-13 cells; RT-PCR, Western blot; XRCC5 siRNA rescue experiment; nude mouse xenograft model","journal":"Anti-cancer drugs","confidence":"Medium","confidence_rationale":"Tier 2 — overexpression and siRNA rescue with multiple readouts, single lab","pmids":["37948318"],"is_preprint":false},{"year":2024,"finding":"Spinal Calcrl+ neurons function as projection neurons that amplify mechanical itch signaling; chemogenetic activation induces mechanical itch sensitization, chemogenetic inhibition alleviates it in chronic itch models, and chronic itch enhances intrinsic excitability and Aβ-fiber-evoked excitatory synaptic input to Calcrl+ neurons with reduced inhibitory input.","method":"Chemogenetic (DREADD) manipulation, behavioral tests, electrophysiology, morphological assays in chronic itch mouse models","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — chemogenetic gain/loss-of-function with electrophysiological and behavioral readouts, single lab","pmids":["41248150"],"is_preprint":false},{"year":2025,"finding":"CALCRL (as core component of the CGRP receptor) is upregulated in AD hippocampus; pharmacological blockade with rimegepant reduces Aβ1-42 oligomer-induced neuronal death and glial inflammation via HDAC11 inhibition, which enhances LXRβ acetylation and ABCA1 expression, reprogramming neuronal lipid metabolism.","method":"5×FAD mouse model; Calca knockout; rimegepant pharmacological blockade; HDAC11/LXRβ/ABCA1 mechanistic pathway analysis; neurobehavioral and neuropathological assays","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 2 — mechanistic pathway identified but preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.10.17.683079"],"is_preprint":true},{"year":2025,"finding":"De novo designed miniprotein antagonists bind CGRPR (CLR/CALCRL + RAMP1) with high affinity; cryo-EM structures confirm atomic-level agreement between designed and experimentally determined structures, establishing precise conformational control of receptor function.","method":"Computational de novo protein design; cryo-electron microscopy structure determination; high-throughput receptor diversion microscopy screen","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 — cryo-EM structural validation of designed antagonist-receptor complex, preprint","pmids":["bio_10.1101_2025.03.23.644666"],"is_preprint":true}],"current_model":"CALCRL (CLR) is a class B GPCR that requires heterodimerization with one of three receptor activity-modifying proteins (RAMPs) to reach the cell surface with full ligand-binding competence: CALCRL/RAMP1 functions as a CGRP receptor (signaling via cAMP, G proteins, and β-arrestin recruitment), while CALCRL/RAMP2 and CALCRL/RAMP3 function as adrenomedullin receptors; the ligand-binding specificity is determined by the RAMP partner, N-glycosylation of CALCRL at Asn123 is required for proper receptor conformation and ligand binding, and the receptor complex mediates downstream effects including vasodilation, angiogenesis, hematopoietic stem cell maintenance, protection from apoptosis, and—when dysregulated—leukemic stem cell survival and chemotherapy resistance."},"narrative":{"teleology":[{"year":1999,"claim":"Establishing that CALCRL requires a RAMP co-receptor for ligand recognition resolved how a single orphan GPCR could serve as both a CGRP and an adrenomedullin receptor depending on its RAMP partner.","evidence":"Co-transfection of RAMP2/CRLR in HeLa and HEK293 cells showing cAMP response to adrenomedullin; extended by HEK293 co-expression with RAMP1/2/3 demonstrating CGRP vs. AM selectivity","pmids":["10217420","10781016"],"confidence":"High","gaps":["Structural basis of RAMP-determined ligand selectivity was not yet resolved","Stoichiometry of the CALCRL-RAMP complex at the cell surface was unknown"]},{"year":2001,"claim":"Site-directed mutagenesis identified Asn123 N-glycosylation as essential for CALCRL ligand binding and surface transport, demonstrating that post-translational modification — not just RAMP identity — governs receptor competence.","evidence":"Mutagenesis of N-glycosylation sites combined with radioligand binding and FACS in HEK293 and CHO-K1 cells; antisense knockdown in cardiomyocytes confirming CRLR is essential for AM signaling","pmids":["11389975","11854283","11754972"],"confidence":"High","gaps":["Contribution of individual RAMP glycosylation sites to heterodimer stability was not yet tested","Whether glycosylation defects affect RAMP-specific pharmacology was unresolved"]},{"year":2003,"claim":"Discovery of HIF-1α-dependent transcriptional regulation of CALCRL through a promoter HRE connected the receptor to hypoxia-responsive gene programs, explaining its upregulation in ischemic tissues.","evidence":"Promoter cloning, HRE mutagenesis, and reporter assays in primary microvascular endothelial cells; RAMP3 mutagenesis in Xenopus oocytes confirming conserved cysteines are required for AM binding","pmids":["12824306","12939163"],"confidence":"High","gaps":["Whether hypoxia-induced CALCRL upregulation alters RAMP stoichiometry and thus ligand selectivity in vivo was untested","Post-transcriptional regulation of CALCRL protein under acute hypoxia was not yet characterized"]},{"year":2005,"claim":"Functional studies extended CALCRL biology into cardioprotection and post-transcriptional regulation, showing CGRP–CALCRL/RAMP1 signaling protects cardiomyoblasts from apoptosis and that acute hypoxia increases CALCRL protein without mRNA changes.","evidence":"Apoptosis assays with CGRP(8-37) blockade in H9c2 cells; Western blot vs. RT-PCR under acute hypoxia in coronary artery smooth muscle cells and chronic hypoxia rat model","pmids":["16242145","15567147"],"confidence":"Medium","gaps":["Mechanism of post-transcriptional CALCRL stabilization under acute hypoxia was not identified","Downstream effectors of CGRP-mediated anti-apoptosis beyond Bcl-2/Bax were unknown"]},{"year":2007,"claim":"BRET-based biophysical studies demonstrated that CALCRL and RAMP1 form selective heterodimers (not random oligomers) and that G protein and β-arrestin recruitment require the intact heterodimer, establishing the quaternary signaling unit.","evidence":"BRET titration assays with CRLR-Rluc and RAMP1-GFP fusions in living cells combined with cAMP and radioligand binding","pmids":["17503773"],"confidence":"High","gaps":["Whether CALCRL homodimers have independent signaling capacity was untested","Structural basis of heterodimer selectivity was unknown at atomic resolution"]},{"year":2016,"claim":"Genetic epistasis in RAMP and Calcrl knockout mice resolved the in vivo receptor pharmacology of vasodepressor responses, showing AM hypotension acts primarily through CLR/RAMP1 with contributions from CLR/RAMP2 and CLR/RAMP3.","evidence":"Blood pressure measurement in Ramp1−/−, Ramp2+/−, Ramp3−/−, double-KO, and Calcrl+/− mice with intravenous ligand injection","pmids":["27940069"],"confidence":"High","gaps":["Tissue-specific RAMP expression patterns driving organ-level AM vs. CGRP responses were not mapped","Whether compensatory RAMP switching occurs in single-RAMP knockout animals was unresolved"]},{"year":2019,"claim":"Multiple studies converged to establish CALCRL as a functional dependency in AML and a regulator of normal hematopoietic stem cells: CRISPR knockout impaired leukemic colony formation, CGRP antagonism reduced leukemic burden, RAMP1 deficiency compromised stress hematopoiesis, and RAMP2 haploinsufficiency caused retinal ganglion cell death.","evidence":"CRISPR-Cas9 KO in AML lines; olcegepant antagonism in mouse AML model; Ramp1−/− BM transplantation and ROS/apoptosis assays; Ramp2+/− mouse retinal phenotyping","pmids":["31182782","31756985","30674976","31000793"],"confidence":"Medium","gaps":["Whether CALCRL dependency in AML is ligand-driven or constitutive was unclear","The relative contribution of CGRP vs. AM signaling arms to leukemic stem cell maintenance was unresolved","Retinal phenotype confirmation in CALCRL-specific conditional knockouts was lacking"]},{"year":2020,"claim":"Crystal structure of erenumab bound to the CGRP receptor revealed that the antibody's CDR-H3 loop inserts into the CLR–RAMP1 interface, providing the first atomic-resolution view of the ligand-binding site and validating the heterodimer interface as a druggable target.","evidence":"Crystal structure of erenumab-CGRPR complex","pmids":["32049005"],"confidence":"High","gaps":["Full-length active-state structure of CALCRL/RAMP1 with CGRP peptide and G protein was not yet determined","Structural basis of RAMP2/3-dependent AM selectivity remained unresolved"]},{"year":2021,"claim":"Mechanistic dissection in patient-derived xenograft models showed the ADM–CALCRL axis drives leukemic stem cell maintenance via E2F1/BCL2-dependent cell cycle progression, DNA repair, and mitochondrial OxPHOS, positioning CALCRL as a chemoresistance node.","evidence":"CALCRL knockdown in PDX models with cell cycle, DNA repair, OxPHOS, and LSC frequency assays post-cytarabine","pmids":["33462236"],"confidence":"High","gaps":["Direct transcriptional targets of CALCRL-E2F1 axis in LSCs were not comprehensively mapped","Whether CALCRL inhibition synergizes with BCL2 inhibitors (venetoclax) was untested"]},{"year":2023,"claim":"CALCRL overexpression was shown to confer daunorubicin resistance through XRCC5 upregulation and AKT/PKCε phosphorylation, with XRCC5 knockdown restoring drug sensitivity — identifying a specific DNA repair effector downstream of CALCRL.","evidence":"CALCRL overexpression and XRCC5 siRNA rescue in HL-60 and Molm-13 cells; nude mouse xenograft model","pmids":["37948318"],"confidence":"Medium","gaps":["Whether CALCRL regulates XRCC5 transcriptionally or post-transcriptionally was not determined","Generalizability to non-AML cancers was untested"]},{"year":2024,"claim":"Identification of an HSF1-bound shear stress-responsive enhancer at rs880890 controlling CALCRL expression in endothelial cells connected the receptor to flow-dependent vascular homeostasis and downstream eNOS/NO signaling.","evidence":"CRISPR enhancer deletion, siRNA knockdown, ATAC-seq, ChIP-qPCR, functional assays in human aortic endothelial cells","pmids":["38602103"],"confidence":"High","gaps":["In vivo validation of the enhancer's contribution to CALCRL expression in shear-exposed vasculature was not performed","Whether this enhancer operates in non-endothelial CALCRL-expressing cell types is unknown"]},{"year":null,"claim":"Key unresolved questions include the structural basis of RAMP2/3-dependent adrenomedullin selectivity at atomic resolution, whether CALCRL signals constitutively in the absence of ligand in disease contexts such as AML, and the therapeutic window for CALCRL inhibition given its dual roles in normal hematopoiesis and leukemic stem cell survival.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full active-state structure of CALCRL/RAMP2 or CALCRL/RAMP3 with G protein","Ligand-independent vs. ligand-dependent CALCRL activity in AML not discriminated","Conditional CALCRL knockout models in adult hematopoietic system not reported"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,1,5,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,5]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,3,5,10]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,5,6,9,14]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[12,13,14,19]},{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[11]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[8,18]}],"complexes":["CLR/RAMP1 (CGRP receptor)","CLR/RAMP2 (AM1 receptor)","CLR/RAMP3 (AM2 receptor)"],"partners":["RAMP1","RAMP2","RAMP3","XRCC5","HSF1","HIF1A"],"other_free_text":[]},"mechanistic_narrative":"CALCRL (calcitonin receptor-like receptor, CLR) is a class B G protein-coupled receptor whose ligand specificity is dictated by obligate heterodimerization with receptor activity-modifying proteins (RAMPs): CALCRL/RAMP1 forms the CGRP receptor, while CALCRL/RAMP2 and CALCRL/RAMP3 constitute adrenomedullin receptors, each coupling to Gαs-mediated cAMP production, G protein recruitment, and β-arrestin signaling [PMID:10781016, PMID:17503773]. N-glycosylation of CALCRL at Asn123 is required for proper receptor conformation, cell-surface transport, and ligand binding [PMID:11389975], and the CALCRL promoter contains a functional HIF-1α-responsive element that upregulates expression under hypoxia [PMID:12824306]. In the vasculature, CALCRL mediates vasodilation, endothelial angiogenesis, eNOS activation, and retinal ganglion cell survival through RAMP-dependent signaling [PMID:27940069, PMID:14712479, PMID:38602103, PMID:31000793]; in hematopoiesis, the CGRP–CALCRL/RAMP1 axis supports stress-induced hematopoietic stem cell function [PMID:30674976], while in acute myeloid leukemia the ADM–CALCRL axis drives leukemic stem cell maintenance, cell cycle progression, DNA repair, OxPHOS, and chemotherapy resistance through E2F1/BCL2- and XRCC5/AKT-dependent pathways [PMID:33462236, PMID:37948318]."},"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. Activates cAMP-dependent pathway (PubMed:32296767, PubMed:8626685)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q16602/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CALCRL","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/CALCRL","total_profiled":1310},"omim":[{"mim_id":"618773","title":"LYMPHATIC MALFORMATION 8; LMPHM8","url":"https://www.omim.org/entry/618773"},{"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":"153100","title":"LYMPHATIC MALFORMATION 1; 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 RAMP2/CRLR complex is a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells.","date":"1999","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/10217420","citation_count":113,"is_preprint":false},{"pmid":"10781016","id":"PMC_10781016","title":"CGRP and adrenomedullin binding correlates with transcript levels for calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) in rat tissues.","date":"2000","source":"British journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/10781016","citation_count":112,"is_preprint":false},{"pmid":"14712479","id":"PMC_14712479","title":"Effects of adrenomedullin on endothelial cells in the multistep process of angiogenesis: involvement of CRLR/RAMP2 and CRLR/RAMP3 receptors.","date":"2004","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/14712479","citation_count":110,"is_preprint":false},{"pmid":"12824306","id":"PMC_12824306","title":"Transcriptional regulation of the CRLR gene in human microvascular endothelial cells by hypoxia.","date":"2003","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/12824306","citation_count":66,"is_preprint":false},{"pmid":"12086988","id":"PMC_12086988","title":"Comparison of the expression of calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) with CGRP and adrenomedullin binding in cell lines.","date":"2002","source":"British journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/12086988","citation_count":64,"is_preprint":false},{"pmid":"33462236","id":"PMC_33462236","title":"Adrenomedullin-CALCRL axis controls relapse-initiating drug tolerant acute myeloid leukemia 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correlation of RAMP mRNA with binding in rat tissues\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — multiple orthogonal methods (binding + functional assay + tissue correlation), independently replicated\",\n      \"pmids\": [\"10781016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CRLR cell-surface expression does not require heterodimer assembly with RAMPs; RAMP2 and RAMP3 reach the plasma membrane via N-glycosylation-dependent mechanisms, while RAMP1 (non-glycosylated) requires heterodimerization with CRLR for plasma membrane targeting.\",\n      \"method\": \"Xenopus oocyte expression system with quantitative cell-surface binding assay; N-glycosylation site mutagenesis of RAMP1\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution in Xenopus oocytes with mutagenesis and quantitative surface detection\",\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; loss of glycosylation at this site impairs cell-surface transport and receptor conformation without affecting expression of N66Q or N118Q mutants.\",\n      \"method\": \"Site-directed mutagenesis of N-glycosylation sites, radioligand binding, FACS analysis in HEK293 EBNA and CHO-K1 cells\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis combined with binding and surface expression assays\",\n      \"pmids\": [\"11389975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"N-glycosylation and conserved cysteine residues in RAMP3 are critical for functional expression of the CRLR/RAMP3 adrenomedullin receptor; elimination of all N-glycans reduces AM binding, and mutation of all six cysteines abolishes adrenomedullin binding.\",\n      \"method\": \"Xenopus oocyte expression system with [125I]AM radioligand binding, N-glycosylation site and cysteine mutagenesis\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution with systematic mutagenesis and binding assay\",\n      \"pmids\": [\"12939163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CRLR and RAMP1 form selective heterodimers at the cell surface (not random oligomers); both CRLR and RAMP1 can also form homodimers. CRLR recruits G proteins and β-arrestin upon CGRP stimulation only in the presence of RAMP1.\",\n      \"method\": \"BRET titration assays in living cells; radioligand binding; cAMP production assays with CRLR-Rluc and RAMP1-GFP fusion proteins\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal BRET methods plus functional assays in living cells\",\n      \"pmids\": [\"17503773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Adrenomedullin signaling in rat cardiomyocytes requires CRLR and RAMP2 to constitute functional AM receptors; overexpression of CRLR or RAMP2 potentiates AM-induced CRE-luciferase (cAMP) signaling, and CRLR antisense abolishes AM response.\",\n      \"method\": \"Transient transfection of cardiomyocytes with CRE-luciferase reporter; CRLR antisense and receptor antagonist CGRP(8-37) blockade\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — functional reporter assay with antisense knockdown and pharmacological antagonism\",\n      \"pmids\": [\"11754972\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The human CRLR gene promoter contains a functional hypoxia-response element (HRE) that is activated by HIF-1α under hypoxia; site-directed mutagenesis of the HRE abolishes hypoxia-induced promoter activity in primary microvascular endothelial cells.\",\n      \"method\": \"5'-RACE, promoter cloning, reporter gene assays, site-directed mutagenesis of HRE, semi-quantitative RT-PCR under hypoxic conditions\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis of HRE combined with reporter assays and mRNA quantification\",\n      \"pmids\": [\"12824306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CRLR/RAMP2 and CRLR/RAMP3 receptors mediate adrenomedullin-induced HUVEC migration, invasion, and differentiation into cord-like structures during angiogenesis; AM acts independently of VEGF in capillary tube formation.\",\n      \"method\": \"HUVEC migration and invasion assays, Matrigel differentiation assay, blocking antibodies and receptor-specific pharmacology\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple cellular assays with pharmacological blockade, single lab\",\n      \"pmids\": [\"14712479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CGRP exerts antiapoptotic effects in H9c2 cardiomyoblasts specifically through the RAMP1/CRLR complex, as shown by CGRP(8-37) antagonist blockade; RAMP1 expression is upregulated by CGRP and CGRP prevents oxidative stress-induced Bcl-2 decrease and Bax increase.\",\n      \"method\": \"MTT assays, caspase-3 activation, DNA fragmentation, RT-PCR, dot blot; CGRP(8-37) antagonist and adrenomedullin comparison in H9c2 cells\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal assays with pharmacological blockade, single lab\",\n      \"pmids\": [\"16242145\"],\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 interface between CLR (CALCRL) and RAMP1, directly blocking ligand binding by contacting residues specific to both CLR and RAMP1.\",\n      \"method\": \"Crystal structure determination of erenumab-CGRPR complex\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation of direct ligand-blocking mechanism\",\n      \"pmids\": [\"32049005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The hypotensive response to adrenomedullin is primarily mediated through CLR/RAMP1, with contributions from CLR/RAMP2 and CLR/RAMP3; CGRP hypotension is predominantly through CLR/RAMP1. Genetic reduction of Calcrl attenuates the hypotensive response to both AM and CGRP in vivo.\",\n      \"method\": \"Blood pressure measurement in Ramp1-/-, Ramp2+/-, Ramp3-/-, Ramp1-/-/Ramp3-/- double-KO, and Calcrl+/- mice with intravenous AM and CGRP injection\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis in multiple KO/KD mouse models with defined cardiovascular phenotype\",\n      \"pmids\": [\"27940069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CALCRL knockdown by CRISPR-Cas9 significantly impairs colony formation in human myeloid leukemia cell lines, establishing a functional role for CALCRL in leukemic cell growth.\",\n      \"method\": \"CRISPR-Cas9 knockout in human AML cell lines with colony formation assay\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotype, single lab\",\n      \"pmids\": [\"31182782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The CGRP-CALCRL/RAMP1 axis protects AML cells from chemotherapy-induced apoptosis; CGRP antagonist olcegepant increases differentiation, reduces leukemic burden, and decreases stem cell properties in a mouse AML model.\",\n      \"method\": \"Apoptosis assays in AML cell lines and primary samples; CGRP antagonist (olcegepant) treatment; C57BL/6 mouse AML model with in vivo leukemic burden and stem cell assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional assays with pharmacological antagonism and in vivo model, 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; CALCRL knockdown decreases leukemic stem cell frequency and sensitizes to cytarabine in patient-derived xenograft models.\",\n      \"method\": \"CALCRL knockdown in patient-derived xenograft models; cell cycle, DNA repair, and OxPHOS functional assays; in vivo LSC frequency assays post-chemotherapy\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal mechanistic assays plus in vivo patient-derived xenograft models\",\n      \"pmids\": [\"33462236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CGRP-CALCRL/RAMP1 signaling is required for stress-induced hematopoiesis; Ramp1-deficient mice show decreased bone marrow repopulation capacity, reduced proliferation, enhanced ROS production, and increased apoptosis under proliferative stress.\",\n      \"method\": \"Ramp1-/- mouse model; BM transplantation/repopulation assay; ROS measurement; apoptosis assay; CGRP administration experiments\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO model with multiple functional readouts, 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 AM-RAMP2/CRLR-cAMP signaling; ablation of one Ramp2 allele leads to cAMP reduction and retinal ganglion cell death in mice, establishing that the RAMP2/CRLR-cAMP axis is essential for retinal ganglion cell survival.\",\n      \"method\": \"Heterozygous Ramp2 knockout mouse model; transfection of mutant RAMP2 constructs; cAMP measurement; retinal ganglion cell death assay\",\n      \"journal\": \"Genetics in medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse model combined with functional cAMP assay and cell death quantification\",\n      \"pmids\": [\"31000793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Acute hypoxia in coronary artery smooth muscle cells induces a rapid increase in CRLR protein independently of changes in CRLR mRNA, indicating a post-transcriptional regulatory mechanism; chronic hypoxia in rats enhances both mRNA and protein of CRLR and all three RAMPs in cardiac ventricles.\",\n      \"method\": \"Western blotting and RT-PCR in human coronary artery smooth muscle cells under acute hypoxia; chronic hypobaric hypoxia rat model\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — two complementary experimental models with protein and mRNA quantification\",\n      \"pmids\": [\"15567147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CALCRL expression in endothelial cells is regulated by an HSF1-bound shear stress-responsive enhancer at rs880890; CRISPR deletion of this enhancer downregulates CALCRL expression, and CALCRL knockdown reduces eNOS, apelin, angiopoietin, prostaglandin, and EDN1 signaling, decreasing cell proliferation, tube formation, and NO production.\",\n      \"method\": \"CRISPR enhancer deletion, siRNA knockdown, ATAC-seq, ChIP-qPCR, electromobility shift assay, luciferase reporter assay, functional proliferation and tube formation assays in human aortic endothelial cells\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — multiple orthogonal epigenomic, genetic, and functional methods in a single study\",\n      \"pmids\": [\"38602103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CALCRL overexpression in AML cells confers resistance to daunorubicin through upregulation of XRCC5 and PDK1, leading to increased AKT/PKCε phosphorylation; XRCC5 siRNA in CALCRL-overexpressing cells restores drug sensitivity and increases apoptosis.\",\n      \"method\": \"CALCRL overexpression constructs in HL-60 and Molm-13 cells; RT-PCR, Western blot; XRCC5 siRNA rescue experiment; nude mouse xenograft model\",\n      \"journal\": \"Anti-cancer drugs\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — overexpression and siRNA rescue with multiple readouts, single lab\",\n      \"pmids\": [\"37948318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Spinal Calcrl+ neurons function as projection neurons that amplify mechanical itch signaling; chemogenetic activation induces mechanical itch sensitization, chemogenetic inhibition alleviates it in chronic itch models, and chronic itch enhances intrinsic excitability and Aβ-fiber-evoked excitatory synaptic input to Calcrl+ neurons with reduced inhibitory input.\",\n      \"method\": \"Chemogenetic (DREADD) manipulation, behavioral tests, electrophysiology, morphological assays in chronic itch mouse models\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — chemogenetic gain/loss-of-function with electrophysiological and behavioral readouts, single lab\",\n      \"pmids\": [\"41248150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CALCRL (as core component of the CGRP receptor) is upregulated in AD hippocampus; pharmacological blockade with rimegepant reduces Aβ1-42 oligomer-induced neuronal death and glial inflammation via HDAC11 inhibition, which enhances LXRβ acetylation and ABCA1 expression, reprogramming neuronal lipid metabolism.\",\n      \"method\": \"5×FAD mouse model; Calca knockout; rimegepant pharmacological blockade; HDAC11/LXRβ/ABCA1 mechanistic pathway analysis; neurobehavioral and neuropathological assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic pathway identified but preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.10.17.683079\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"De novo designed miniprotein antagonists bind CGRPR (CLR/CALCRL + RAMP1) with high affinity; cryo-EM structures confirm atomic-level agreement between designed and experimentally determined structures, establishing precise conformational control of receptor function.\",\n      \"method\": \"Computational de novo protein design; cryo-electron microscopy structure determination; high-throughput receptor diversion microscopy screen\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structural validation of designed antagonist-receptor complex, preprint\",\n      \"pmids\": [\"bio_10.1101_2025.03.23.644666\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CALCRL (CLR) is a class B GPCR that requires heterodimerization with one of three receptor activity-modifying proteins (RAMPs) to reach the cell surface with full ligand-binding competence: CALCRL/RAMP1 functions as a CGRP receptor (signaling via cAMP, G proteins, and β-arrestin recruitment), while CALCRL/RAMP2 and CALCRL/RAMP3 function as adrenomedullin receptors; the ligand-binding specificity is determined by the RAMP partner, N-glycosylation of CALCRL at Asn123 is required for proper receptor conformation and ligand binding, and the receptor complex mediates downstream effects including vasodilation, angiogenesis, hematopoietic stem cell maintenance, protection from apoptosis, and—when dysregulated—leukemic stem cell survival and chemotherapy resistance.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CALCRL (calcitonin receptor-like receptor, CLR) is a class B G protein-coupled receptor whose ligand specificity is dictated by obligate heterodimerization with receptor activity-modifying proteins (RAMPs): CALCRL/RAMP1 forms the CGRP receptor, while CALCRL/RAMP2 and CALCRL/RAMP3 constitute adrenomedullin receptors, each coupling to Gαs-mediated cAMP production, G protein recruitment, and β-arrestin signaling [PMID:10781016, PMID:17503773]. N-glycosylation of CALCRL at Asn123 is required for proper receptor conformation, cell-surface transport, and ligand binding [PMID:11389975], and the CALCRL promoter contains a functional HIF-1α-responsive element that upregulates expression under hypoxia [PMID:12824306]. In the vasculature, CALCRL mediates vasodilation, endothelial angiogenesis, eNOS activation, and retinal ganglion cell survival through RAMP-dependent signaling [PMID:27940069, PMID:14712479, PMID:38602103, PMID:31000793]; in hematopoiesis, the CGRP–CALCRL/RAMP1 axis supports stress-induced hematopoietic stem cell function [PMID:30674976], while in acute myeloid leukemia the ADM–CALCRL axis drives leukemic stem cell maintenance, cell cycle progression, DNA repair, OxPHOS, and chemotherapy resistance through E2F1/BCL2- and XRCC5/AKT-dependent pathways [PMID:33462236, PMID:37948318].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Establishing that CALCRL requires a RAMP co-receptor for ligand recognition resolved how a single orphan GPCR could serve as both a CGRP and an adrenomedullin receptor depending on its RAMP partner.\",\n      \"evidence\": \"Co-transfection of RAMP2/CRLR in HeLa and HEK293 cells showing cAMP response to adrenomedullin; extended by HEK293 co-expression with RAMP1/2/3 demonstrating CGRP vs. AM selectivity\",\n      \"pmids\": [\"10217420\", \"10781016\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of RAMP-determined ligand selectivity was not yet resolved\", \"Stoichiometry of the CALCRL-RAMP complex at the cell surface was unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Site-directed mutagenesis identified Asn123 N-glycosylation as essential for CALCRL ligand binding and surface transport, demonstrating that post-translational modification — not just RAMP identity — governs receptor competence.\",\n      \"evidence\": \"Mutagenesis of N-glycosylation sites combined with radioligand binding and FACS in HEK293 and CHO-K1 cells; antisense knockdown in cardiomyocytes confirming CRLR is essential for AM signaling\",\n      \"pmids\": [\"11389975\", \"11854283\", \"11754972\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Contribution of individual RAMP glycosylation sites to heterodimer stability was not yet tested\", \"Whether glycosylation defects affect RAMP-specific pharmacology was unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Discovery of HIF-1α-dependent transcriptional regulation of CALCRL through a promoter HRE connected the receptor to hypoxia-responsive gene programs, explaining its upregulation in ischemic tissues.\",\n      \"evidence\": \"Promoter cloning, HRE mutagenesis, and reporter assays in primary microvascular endothelial cells; RAMP3 mutagenesis in Xenopus oocytes confirming conserved cysteines are required for AM binding\",\n      \"pmids\": [\"12824306\", \"12939163\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether hypoxia-induced CALCRL upregulation alters RAMP stoichiometry and thus ligand selectivity in vivo was untested\", \"Post-transcriptional regulation of CALCRL protein under acute hypoxia was not yet characterized\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Functional studies extended CALCRL biology into cardioprotection and post-transcriptional regulation, showing CGRP–CALCRL/RAMP1 signaling protects cardiomyoblasts from apoptosis and that acute hypoxia increases CALCRL protein without mRNA changes.\",\n      \"evidence\": \"Apoptosis assays with CGRP(8-37) blockade in H9c2 cells; Western blot vs. RT-PCR under acute hypoxia in coronary artery smooth muscle cells and chronic hypoxia rat model\",\n      \"pmids\": [\"16242145\", \"15567147\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of post-transcriptional CALCRL stabilization under acute hypoxia was not identified\", \"Downstream effectors of CGRP-mediated anti-apoptosis beyond Bcl-2/Bax were unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"BRET-based biophysical studies demonstrated that CALCRL and RAMP1 form selective heterodimers (not random oligomers) and that G protein and β-arrestin recruitment require the intact heterodimer, establishing the quaternary signaling unit.\",\n      \"evidence\": \"BRET titration assays with CRLR-Rluc and RAMP1-GFP fusions in living cells combined with cAMP and radioligand binding\",\n      \"pmids\": [\"17503773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CALCRL homodimers have independent signaling capacity was untested\", \"Structural basis of heterodimer selectivity was unknown at atomic resolution\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Genetic epistasis in RAMP and Calcrl knockout mice resolved the in vivo receptor pharmacology of vasodepressor responses, showing AM hypotension acts primarily through CLR/RAMP1 with contributions from CLR/RAMP2 and CLR/RAMP3.\",\n      \"evidence\": \"Blood pressure measurement in Ramp1−/−, Ramp2+/−, Ramp3−/−, double-KO, and Calcrl+/− mice with intravenous ligand injection\",\n      \"pmids\": [\"27940069\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific RAMP expression patterns driving organ-level AM vs. CGRP responses were not mapped\", \"Whether compensatory RAMP switching occurs in single-RAMP knockout animals was unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Multiple studies converged to establish CALCRL as a functional dependency in AML and a regulator of normal hematopoietic stem cells: CRISPR knockout impaired leukemic colony formation, CGRP antagonism reduced leukemic burden, RAMP1 deficiency compromised stress hematopoiesis, and RAMP2 haploinsufficiency caused retinal ganglion cell death.\",\n      \"evidence\": \"CRISPR-Cas9 KO in AML lines; olcegepant antagonism in mouse AML model; Ramp1−/− BM transplantation and ROS/apoptosis assays; Ramp2+/− mouse retinal phenotyping\",\n      \"pmids\": [\"31182782\", \"31756985\", \"30674976\", \"31000793\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CALCRL dependency in AML is ligand-driven or constitutive was unclear\", \"The relative contribution of CGRP vs. AM signaling arms to leukemic stem cell maintenance was unresolved\", \"Retinal phenotype confirmation in CALCRL-specific conditional knockouts was lacking\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Crystal structure of erenumab bound to the CGRP receptor revealed that the antibody's CDR-H3 loop inserts into the CLR–RAMP1 interface, providing the first atomic-resolution view of the ligand-binding site and validating the heterodimer interface as a druggable target.\",\n      \"evidence\": \"Crystal structure of erenumab-CGRPR complex\",\n      \"pmids\": [\"32049005\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length active-state structure of CALCRL/RAMP1 with CGRP peptide and G protein was not yet determined\", \"Structural basis of RAMP2/3-dependent AM selectivity remained unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mechanistic dissection in patient-derived xenograft models showed the ADM–CALCRL axis drives leukemic stem cell maintenance via E2F1/BCL2-dependent cell cycle progression, DNA repair, and mitochondrial OxPHOS, positioning CALCRL as a chemoresistance node.\",\n      \"evidence\": \"CALCRL knockdown in PDX models with cell cycle, DNA repair, OxPHOS, and LSC frequency assays post-cytarabine\",\n      \"pmids\": [\"33462236\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct transcriptional targets of CALCRL-E2F1 axis in LSCs were not comprehensively mapped\", \"Whether CALCRL inhibition synergizes with BCL2 inhibitors (venetoclax) was untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"CALCRL overexpression was shown to confer daunorubicin resistance through XRCC5 upregulation and AKT/PKCε phosphorylation, with XRCC5 knockdown restoring drug sensitivity — identifying a specific DNA repair effector downstream of CALCRL.\",\n      \"evidence\": \"CALCRL overexpression and XRCC5 siRNA rescue in HL-60 and Molm-13 cells; nude mouse xenograft model\",\n      \"pmids\": [\"37948318\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CALCRL regulates XRCC5 transcriptionally or post-transcriptionally was not determined\", \"Generalizability to non-AML cancers was untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identification of an HSF1-bound shear stress-responsive enhancer at rs880890 controlling CALCRL expression in endothelial cells connected the receptor to flow-dependent vascular homeostasis and downstream eNOS/NO signaling.\",\n      \"evidence\": \"CRISPR enhancer deletion, siRNA knockdown, ATAC-seq, ChIP-qPCR, functional assays in human aortic endothelial cells\",\n      \"pmids\": [\"38602103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo validation of the enhancer's contribution to CALCRL expression in shear-exposed vasculature was not performed\", \"Whether this enhancer operates in non-endothelial CALCRL-expressing cell types is unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of RAMP2/3-dependent adrenomedullin selectivity at atomic resolution, whether CALCRL signals constitutively in the absence of ligand in disease contexts such as AML, and the therapeutic window for CALCRL inhibition given its dual roles in normal hematopoiesis and leukemic stem cell survival.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full active-state structure of CALCRL/RAMP2 or CALCRL/RAMP3 with G protein\", \"Ligand-independent vs. ligand-dependent CALCRL activity in AML not discriminated\", \"Conditional CALCRL knockout models in adult hematopoietic system not reported\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 5, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 3, 5, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 5, 6, 9, 14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [12, 13, 14, 19]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [8, 18]}\n    ],\n    \"complexes\": [\n      \"CLR/RAMP1 (CGRP receptor)\",\n      \"CLR/RAMP2 (AM1 receptor)\",\n      \"CLR/RAMP3 (AM2 receptor)\"\n    ],\n    \"partners\": [\n      \"RAMP1\",\n      \"RAMP2\",\n      \"RAMP3\",\n      \"XRCC5\",\n      \"HSF1\",\n      \"HIF1A\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}