{"gene":"ADGRD1","run_date":"2026-06-09T22:02:41","timeline":{"discoveries":[{"year":2011,"finding":"GPR133 (ADGRD1) couples to Gs protein and activates the Gs/adenylyl cyclase pathway to elevate cAMP. Neither the N-terminal ectodomain nor cleavage at the GPCR proteolysis site is required for G protein signaling. Gs coupling was verified by Gαs siRNA knockdown, Gαs overexpression, chimeric Gq(s4) co-expression routing activity to PLC/IP pathway, and a transmembrane-domain missense mutation that abolished receptor activity without altering cell surface expression.","method":"siRNA knockdown of Gαs, overexpression of Gαs, chimeric G protein co-expression, missense mutagenesis, cAMP assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (knockdown, overexpression, chimeric protein, mutagenesis) in a single rigorous study, replicated across conditions","pmids":["22025619"],"is_preprint":false},{"year":2014,"finding":"A short peptide sequence within the ectodomain of GPR133 (termed the Stachel sequence) functions as a tethered agonist; upon structural changes in the ectodomain, this intramolecular agonist is exposed to the seven-transmembrane domain to trigger G protein activation. The Stachel sequence shows high receptor specificity.","method":"Peptide agonist assay, zebrafish Stachel-mutant genetic rescue, exogenous peptide application in hypomorphic gpr126 mutants","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro peptide assay plus in vivo genetic rescue in zebrafish, replicated across two aGPCRs, independently confirmed by later structural work","pmids":["25533341"],"is_preprint":false},{"year":2016,"finding":"GPR133 knockdown reduces CD133+ glioblastoma stem cell prevalence, tumor cell proliferation, and tumorsphere formation in vitro, and markedly reduces tumor xenograft growth in vivo; the GPR133 knockdown phenotype is rescued by forskolin, indicating signaling is mediated through cAMP. GPR133 mRNA is transcriptionally upregulated by hypoxia in a HIF-1α-dependent manner.","method":"shRNA knockdown, tumorsphere assay, mouse xenograft implantation, forskolin rescue, HIF-1α-dependent transcription analysis","journal":"Oncogenesis","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function with defined cellular and in vivo phenotypes plus pharmacological rescue, replicated in patient-derived GBM cultures and xenograft model","pmids":["27775701"],"is_preprint":false},{"year":2016,"finding":"Functional characterization of naturally occurring ADGRD1 missense variants identified several loss-of-function nsSNPs (A448D, Q600stop, C632fs, A761E, N795K) and one gain-of-function nsSNP (F383S) that significantly increased basal receptor activity.","method":"Site-directed mutagenesis, cAMP functional assay","journal":"BMC genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct mutagenesis and functional cAMP assay in a single lab study","pmids":["27516204"],"is_preprint":false},{"year":2021,"finding":"GPR133 undergoes autoproteolytic cleavage shortly after protein synthesis; the N-terminal fragment (NTF) and C-terminal fragment (CTF) remain noncovalently associated until the receptor is trafficked to the plasma membrane, where NTF-CTF dissociation occurs. Cleavage-competent WT GPR133 generates significantly more cAMP than the uncleavable H543R mutant. A PAR1-CTF/GPR133-NTF proxy system confirmed that thrombin-induced NTF shedding increases intracellular cAMP, supporting a model where NTF dissociation at the plasma membrane promotes receptor activation.","method":"Subcellular fractionation, co-immunoprecipitation, uncleavable point mutant (H543R), PAR1 chimeric proxy system with thrombin cleavage, cAMP assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal approaches (subcellular fractionation, Co-IP, mutagenesis, chimeric proxy system) in patient-derived GBM and HEK293T cells","pmids":["34022221"],"is_preprint":false},{"year":2021,"finding":"Adgrd1 is expressed on oviductal epithelium; female mice lacking Adgrd1 are sterile due to failure to relieve the ampullary-isthmic junction (AIJ) restraining mechanism, causing inappropriate retention of embryos in the oviduct. Post-ovulatory attenuation of tubal fluid flow is dysregulated in Adgrd1-deficient mice. The extracellular protein Plxdc2, displayed on cumulus cells, was identified as an activating ligand for Adgrd1 by a large-scale extracellular protein interaction screen.","method":"Constitutive knockout mice, oviductal fluid flow measurement, large-scale extracellular protein interaction screen (AVEXIS), embryo transit imaging","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — constitutive KO with specific reproductive phenotype, functional fluid-flow assay, and ligand identification by orthogonal protein interaction screen","pmids":["33623007"],"is_preprint":false},{"year":2022,"finding":"Cryo-EM structures of ADGRD1 (and ADGRF1) in complex with Gs protein revealed that the stalk region preceding the first transmembrane helix acts as the tethered agonist by forming extensive interactions with the transmembrane domain; an autoproteolysis-deficient ADGRF1 structure showed a cleavage-independent manner of receptor activation. A conserved cascade of inter-helix interaction cores mediates stalk-induced activation.","method":"Cryo-EM structure determination, mutagenesis, functional signaling assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure with mutagenesis and functional validation, replicated across two aGPCRs in one rigorous study","pmids":["35418679"],"is_preprint":false},{"year":2022,"finding":"Antibodies targeting the N-terminus of GPR133 increase cAMP in a concentration-dependent manner. This effect requires autoproteolytic cleavage: cells expressing the cleavage-deficient H543R mutant did not respond to antibody stimulation. Antibody treatment promotes release of the autoproteolytically cleaved NTF, supporting the model that NTF dissociation promotes receptor activation.","method":"Antibody treatment of HEK293T cells and patient-derived GBM cells, cAMP assay, cleavage-deficient mutant (H543R), immunoprecipitation of NTF from conditioned medium","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional antibody activation assay with cleavage-deficient mutant control and NTF detection, single lab","pmids":["35447113"],"is_preprint":false},{"year":2023,"finding":"PTK7 is an extracellular binding partner of GPR133 in glioblastoma, identified by affinity proteomics. PTK7 binds the autoproteolytically generated NTF of GPR133 and its expression in trans increases GPR133 signaling. This allosteric effect requires GPR133 intramolecular cleavage and PTK7 anchoring in the plasma membrane. PTK7's allosteric action is additive with but topographically distinct from orthosteric Stachel peptide activation.","method":"Affinity proteomics, co-immunoprecipitation, cAMP assay, cleavage-deficient mutant, PTK7 transmembrane-anchoring requirement test, shRNA knockdown in GBM","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — affinity proteomics followed by reciprocal Co-IP, functional cAMP assay, mutagenesis/domain requirement experiments, and in vivo knockdown in GBM, all in a single thorough study","pmids":["37354459"],"is_preprint":false},{"year":2024,"finding":"ESYT1, a Ca2+-dependent mediator of ER-plasma membrane bridge formation, is an intracellular interactor of GPR133 identified by proximity biotinylation proteomics. ESYT1 knockdown/knockout increases GPR133 signaling; overexpression suppresses it without altering plasma membrane GPR133 levels. The interaction requires the Ca2+-sensing C2C domain of ESYT1. Thapsigargin-mediated cytosolic Ca2+ elevation promotes ESYT1-GPR133 dissociation, relieving the signaling-suppressive effect.","method":"Proximity biotinylation proteomics (BioID), co-immunoprecipitation, ESYT1 KD/KO and overexpression, domain mutagenesis (C2C), thapsigargin treatment, cAMP assay, GBM tumor growth assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — proximity proteomics plus reciprocal Co-IP, domain-specific mutagenesis, pharmacological dissociation, multiple orthogonal functional readouts in single comprehensive study","pmids":["38758649"],"is_preprint":false},{"year":2025,"finding":"Constitutive and osteoblast-specific knockouts of Gpr133/Adgrd1 in mice cause reduced cortical bone mass and trabecularization characteristic of osteoporosis, due to impaired osteoblast function and increased osteoclast activity. GPR133/ADGRD1 regulates osteoblast differentiation through a combined mechanism involving PTK7 interaction and mechanical forces (demonstrated by stretch assays in vitro and mechanical loading in vivo). Downstream signaling proceeds via cAMP-dependent activation of the β-catenin pathway. Pharmacological activation with agonist AP-970/43482503 (AP503) enhances osteoblast function and alleviates osteoporosis in ovariectomized mice.","method":"Constitutive and osteoblast-specific knockout mice, in vitro stretch assay, in vivo mechanical loading, cAMP assay, β-catenin pathway analysis, pharmacological agonist treatment, ovariectomy osteoporosis model","journal":"Signal transduction and targeted therapy","confidence":"High","confidence_rationale":"Tier 2 / Strong — tissue-specific KO with defined skeletal phenotype, multiple in vitro and in vivo mechanistic experiments including mechanosensing and pathway analysis","pmids":["40583059"],"is_preprint":false},{"year":2025,"finding":"The small molecule GL64, identified as a selective ADGRD1 agonist, activates ADGRD1 by mimicking the Stachel sequence. ADGRD1 negatively regulates osteoclastogenesis via the cAMP-PKA-NFATC1 signaling pathway, and GL64 administration prevents bone loss in an ovariectomy mouse model.","method":"Small-molecule agonist identification, cAMP assay, PKA and NFATC1 pathway analysis, osteoclast differentiation assay, ovariectomy mouse model","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — small-molecule tool compound with defined mechanism of action (Stachel mimicry), downstream pathway dissection, and in vivo validation","pmids":["40644539"],"is_preprint":false},{"year":2025,"finding":"GPR133 exhibits constitutive self-activation via its Stachel sequence and can activate downstream G13 signaling in addition to Gs. A cryo-EM structure of the GPR133-GAIN-miniGα13 complex was resolved at 3.51 Å, revealing both conserved and distinct features compared to the previously resolved GPR133-CTF-Gs complex.","method":"Cryo-EM structure determination (3.51 Å), in vitro reconstitution of GPR133-GAIN-miniGα13 complex, G13 signaling assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — high-quality cryo-EM structure with in vitro reconstitution, but single lab and newly reported finding without independent replication","pmids":["40570642"],"is_preprint":false},{"year":2024,"finding":"GPR133 upregulation in decidual macrophages (caused by promoter hypomethylation) impairs phagocytic function; GPR133 knockdown in THP-1 macrophages significantly improves phagocytic function.","method":"5-Aza-dC demethylation, siRNA/shRNA knockdown, phagocytosis assay in decidual macrophages and THP-1 cells","journal":"Epigenetics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — functional knockdown with defined cellular phenotype (phagocytosis assay), supported by epigenetic manipulation, single lab","pmids":["38564758"],"is_preprint":false},{"year":2025,"finding":"ADGRD1 promotes differentiation of adipose progenitor cells (APCs) in vitro and in vivo. In an obese mouse model (high-fat diet), gain-of-function and loss-of-function studies validated that ADGRD1 promotes adipogenesis and improves metabolic homeostasis. Transcription factors MEF2D and TCF12 were identified as regulators of ADGRD1 expression.","method":"Single-nucleus sequencing trajectory inference, primary APC differentiation assay, gain- and loss-of-function in HFD mouse model, ChIP-seq and RNA-seq analysis","journal":"Science China. Life sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — in vitro and in vivo gain/loss-of-function with metabolic phenotype, supported by multi-omic transcription factor analysis, single lab","pmids":["39821834"],"is_preprint":false}],"current_model":"ADGRD1/GPR133 is an adhesion GPCR that is autoproteolytically cleaved at its GAIN domain GPS site shortly after synthesis; the NTF and CTF remain noncovalently associated until the receptor reaches the plasma membrane, where NTF dissociation exposes the Stachel tethered-agonist sequence in the stalk region, which inserts into the transmembrane domain to activate primarily Gs (elevating cAMP) and also G13 signaling. Cryo-EM structures have resolved the molecular basis of this stalk–TMD interaction. Receptor activity is positively modulated by the extracellular binding partner PTK7 (which acts allosterically on the NTF) and by mechanical forces, while it is negatively regulated intracellularly by ESYT1 in a Ca2+-dependent manner. Physiologically, ADGRD1 controls oviductal fluid flow and embryo transit, promotes osteoblast differentiation and bone formation via cAMP/β-catenin signaling, inhibits osteoclastogenesis via cAMP-PKA-NFATC1, promotes adipogenesis, and supports glioblastoma stem-cell growth."},"narrative":{"mechanistic_narrative":"ADGRD1 (GPR133) is an adhesion G protein-coupled receptor that transduces extracellular and mechanical cues into intracellular cAMP signaling to control reproductive, skeletal, metabolic, and tumor-cell biology [PMID:22025619, PMID:33623007, PMID:40583059]. It couples to Gs to activate adenylyl cyclase and elevate cAMP, and also engages G13 [PMID:22025619, PMID:40570642]. Activation is governed by a tethered-agonist mechanism: the receptor undergoes autoproteolytic cleavage at its GAIN/GPS site shortly after synthesis, generating an N-terminal fragment (NTF) that stays noncovalently associated with the C-terminal fragment until it dissociates at the plasma membrane, exposing the intramolecular Stachel sequence that inserts into the transmembrane domain to drive signaling [PMID:25533341, PMID:34022221, PMID:35418679]. Cryo-EM structures have resolved how the stalk engages the transmembrane domain through a conserved cascade of inter-helix interaction cores, including a cleavage-independent mode of activation [PMID:35418679, PMID:40570642]. Receptor output is tuned by binding partners: the extracellular protein PTK7 binds the NTF and allosterically potentiates signaling in a cleavage-dependent, membrane-anchored manner topographically distinct from Stachel activation [PMID:37354459], mechanical force enhances activity [PMID:40583059], and the Ca2+-dependent ER-plasma membrane tether ESYT1 binds intracellularly to suppress signaling, an inhibition relieved by cytosolic Ca2+ elevation [PMID:38758649]. Plxdc2 displayed on cumulus cells acts as an activating ligand controlling oviductal fluid flow and embryo transit, loss of which renders female mice sterile [PMID:33623007]. Through cAMP signaling, ADGRD1 promotes osteoblast differentiation and bone formation via the β-catenin pathway and inhibits osteoclastogenesis via cAMP-PKA-NFATC1, with its loss producing osteoporosis-like skeletal defects [PMID:40583059, PMID:40644539]; it also promotes adipogenesis and metabolic homeostasis [PMID:39821834] and supports glioblastoma stem-cell growth, where it is upregulated by HIF-1α-dependent hypoxic transcription [PMID:27775701].","teleology":[{"year":2011,"claim":"Established the core transduction logic of the receptor by showing GPR133 couples to Gs and elevates cAMP independently of its ectodomain or proteolytic cleavage.","evidence":"Gαs knockdown/overexpression, chimeric G protein routing, missense mutagenesis and cAMP assay","pmids":["22025619"],"confidence":"High","gaps":["Did not identify the physiological activating input","Mechanism of receptor activation at the molecular level unresolved"]},{"year":2014,"claim":"Defined the activation mechanism by identifying the Stachel sequence as an intramolecular tethered agonist that engages the seven-transmembrane domain.","evidence":"Peptide agonist assay plus zebrafish Stachel-mutant genetic rescue across two aGPCRs","pmids":["25533341"],"confidence":"High","gaps":["How ectodomain conformational change exposes the Stachel in vivo not resolved","Endogenous triggers of exposure unknown"]},{"year":2016,"claim":"Linked GPR133 to a disease context by showing it sustains glioblastoma stem-cell growth through cAMP and is transcriptionally induced by hypoxia.","evidence":"shRNA knockdown, tumorsphere and xenograft assays, forskolin rescue, HIF-1α-dependent transcription analysis","pmids":["27775701"],"confidence":"High","gaps":["Endogenous activating ligand in GBM not identified","Downstream effectors beyond cAMP not mapped"]},{"year":2016,"claim":"Connected receptor function to natural sequence variation by functionally classifying loss- and gain-of-function ADGRD1 missense variants.","evidence":"Site-directed mutagenesis and cAMP functional assay","pmids":["27516204"],"confidence":"Medium","gaps":["Single-lab functional characterization without in vivo validation","Physiological consequences of variants unknown"]},{"year":2021,"claim":"Resolved how cleavage controls activation by showing NTF-CTF dissociation at the plasma membrane promotes signaling, with cleavage-competent receptor producing more cAMP than the uncleavable mutant.","evidence":"Subcellular fractionation, Co-IP, H543R uncleavable mutant, PAR1-CTF chimeric proxy with thrombin, cAMP assay","pmids":["34022221"],"confidence":"High","gaps":["Physiological driver of NTF dissociation in native tissue unknown","Whether dissociation is required or sufficient for full activation not fully resolved"]},{"year":2021,"claim":"Defined a physiological role and an activating ligand by showing Adgrd1 controls oviductal fluid flow and embryo transit, with Plxdc2 on cumulus cells acting as ligand.","evidence":"Constitutive KO mice, oviductal fluid-flow measurement, AVEXIS interaction screen, embryo transit imaging","pmids":["33623007"],"confidence":"High","gaps":["Signaling pathway downstream of Plxdc2-Adgrd1 in oviduct not dissected","Whether Plxdc2 acts via the Stachel mechanism unknown"]},{"year":2022,"claim":"Provided the structural basis of tethered-agonist activation by resolving cryo-EM structures showing the stalk engages the transmembrane domain through a conserved inter-helix cascade, including cleavage-independent activation.","evidence":"Cryo-EM of ADGRD1/ADGRF1-Gs complexes, mutagenesis, functional signaling assays","pmids":["35418679"],"confidence":"High","gaps":["Structures captured Gs-bound state only","Conformational transition from inactive to active state not visualized"]},{"year":2022,"claim":"Demonstrated that NTF-directed antibodies can pharmacologically activate the receptor in a cleavage-dependent manner, validating NTF dissociation as a druggable activation step.","evidence":"Antibody treatment of HEK293T and GBM cells, cAMP assay, H543R control, NTF immunoprecipitation","pmids":["35447113"],"confidence":"Medium","gaps":["Single-lab study without independent replication","Therapeutic applicability in vivo not tested"]},{"year":2023,"claim":"Identified PTK7 as an extracellular allosteric positive modulator that binds the NTF and potentiates signaling distinctly from orthosteric Stachel activation.","evidence":"Affinity proteomics, reciprocal Co-IP, cAMP assay, cleavage-deficient mutant, PTK7 membrane-anchoring test, GBM knockdown","pmids":["37354459"],"confidence":"High","gaps":["Structural basis of PTK7-NTF interaction unresolved","Whether PTK7 modulation operates in non-GBM tissues not addressed here"]},{"year":2024,"claim":"Revealed intracellular negative regulation by showing ESYT1 binds GPR133 in a Ca2+-dependent manner to suppress signaling, an inhibition relieved by cytosolic Ca2+ elevation.","evidence":"BioID proximity proteomics, reciprocal Co-IP, ESYT1 KD/KO/overexpression, C2C domain mutagenesis, thapsigargin, cAMP and GBM growth assays","pmids":["38758649"],"confidence":"High","gaps":["How ESYT1 binding mechanically suppresses receptor signaling unclear","Physiological Ca2+ stimuli that gate this regulation in vivo unknown"]},{"year":2024,"claim":"Extended receptor function to innate immunity by showing GPR133 upregulation impairs macrophage phagocytosis.","evidence":"5-Aza-dC demethylation, siRNA/shRNA knockdown, phagocytosis assays in decidual and THP-1 macrophages","pmids":["38564758"],"confidence":"Medium","gaps":["Single-lab functional study","Signaling pathway linking GPR133 to phagocytic suppression not defined"]},{"year":2025,"claim":"Established a skeletal role by showing GPR133 promotes osteoblast differentiation and bone formation via mechanical force, PTK7, and cAMP/β-catenin signaling, with a tractable agonist alleviating osteoporosis.","evidence":"Constitutive and osteoblast-specific KO mice, stretch and mechanical loading assays, β-catenin pathway analysis, AP503 agonist in ovariectomy model","pmids":["40583059"],"confidence":"High","gaps":["Molecular link between mechanical force and Stachel/NTF dynamics not resolved","How cAMP feeds into β-catenin mechanistically not fully defined"]},{"year":2025,"claim":"Showed ADGRD1 negatively regulates osteoclastogenesis via cAMP-PKA-NFATC1 and identified a Stachel-mimicking small-molecule agonist that prevents bone loss.","evidence":"GL64 small-molecule agonist, cAMP/PKA/NFATC1 pathway analysis, osteoclast differentiation assay, ovariectomy model","pmids":["40644539"],"confidence":"High","gaps":["Cell-type specificity of GL64 action not fully delineated","Whether osteoblast and osteoclast effects are cell-autonomous not resolved"]},{"year":2025,"claim":"Expanded signaling repertoire by demonstrating G13 coupling and constitutive Stachel-driven self-activation, with a cryo-EM GAIN-miniGα13 structure.","evidence":"Cryo-EM at 3.51 Å, in vitro reconstitution of GPR133-GAIN-miniGα13, G13 signaling assay","pmids":["40570642"],"confidence":"Medium","gaps":["Single-lab structure without independent replication","Physiological contexts requiring G13 versus Gs signaling unknown"]},{"year":2025,"claim":"Demonstrated a metabolic role by showing ADGRD1 promotes adipocyte progenitor differentiation and improves metabolic homeostasis, with MEF2D and TCF12 controlling its expression.","evidence":"Single-nucleus trajectory inference, primary APC differentiation, gain/loss-of-function in HFD mice, ChIP-seq and RNA-seq","pmids":["39821834"],"confidence":"Medium","gaps":["Downstream signaling in adipocytes not connected to known cAMP mechanism","Activating ligand in adipose tissue not identified"]},{"year":null,"claim":"The endogenous activating inputs (ligands versus mechanical force) and the conformational sequence that exposes the Stachel in native tissues remain incompletely defined across the receptor's diverse physiological roles.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model of how tissue-specific ligands, force, and partner proteins converge on Stachel exposure","Inactive-state structure not resolved","Tissue-specific Gs versus G13 bias not mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,1,6,12]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4,5,8]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,6,8,9]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[5,10,14]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[2]}],"complexes":[],"partners":["PTK7","ESYT1","PLXDC2","GNAS","GNA13"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6QNK2","full_name":"Adhesion G-protein coupled receptor D1","aliases":["G-protein coupled receptor 133","G-protein coupled receptor PGR25"],"length_aa":874,"mass_kda":96.5,"function":"Adhesion G-protein coupled receptor (aGPCR) for androgen hormone 5alpha-dihydrotestosterone (5alpha-DHT), also named 17beta-hydroxy-5alpha-androstan-3-one, the most potent hormone among androgens (PubMed:39884271). Also activated by methenolone drug (PubMed:39884271). Ligand binding causes a conformation change that triggers signaling via guanine nucleotide-binding proteins (G proteins) and modulates the activity of downstream effectors, such as adenylate cyclase (PubMed:39884271). ADGRD1 is coupled to G(s) G proteins and mediates activation of adenylate cyclase activity (PubMed:22025619, PubMed:22575658, PubMed:35447113, PubMed:39884271). Acts as a 5alpha-DHT receptor in muscle cells, thereby increasing intracellular cyclic AMP (cAMP) levels and enhancing muscle strength (PubMed:39884271)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q6QNK2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ADGRD1","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ADGRD1","total_profiled":1310},"omim":[{"mim_id":"613639","title":"ADHESION G PROTEIN-COUPLED RECEPTOR D1; ADGRD1","url":"https://www.omim.org/entry/613639"},{"mim_id":"611582","title":"FAMILY WITH SEQUENCE SIMILARITY 12, MEMBER B; FAM12B","url":"https://www.omim.org/entry/611582"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Nuclear speckles","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"heart muscle","ntpm":54.6}],"url":"https://www.proteinatlas.org/search/ADGRD1"},"hgnc":{"alias_symbol":["DKFZp434B1272","PGR25"],"prev_symbol":["GPR133"]},"alphafold":{"accession":"Q6QNK2","domains":[{"cath_id":"2.60.120,2.60.120","chopping":"35-249","consensus_level":"high","plddt":63.9124,"start":35,"end":249},{"cath_id":"2.60.220.50","chopping":"311-556","consensus_level":"medium","plddt":83.3722,"start":311,"end":556},{"cath_id":"1.20.1070.10","chopping":"570-813","consensus_level":"high","plddt":83.2414,"start":570,"end":813}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6QNK2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6QNK2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6QNK2-F1-predicted_aligned_error_v6.png","plddt_mean":71.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ADGRD1","jax_strain_url":"https://www.jax.org/strain/search?query=ADGRD1"},"sequence":{"accession":"Q6QNK2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6QNK2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6QNK2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6QNK2"}},"corpus_meta":[{"pmid":"25533341","id":"PMC_25533341","title":"A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.","date":"2014","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/25533341","citation_count":231,"is_preprint":false},{"pmid":"35418679","id":"PMC_35418679","title":"Structural basis of tethered agonism of the adhesion GPCRs ADGRD1 and ADGRF1.","date":"2022","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/35418679","citation_count":105,"is_preprint":false},{"pmid":"22025619","id":"PMC_22025619","title":"Cell adhesion receptor GPR133 couples to Gs protein.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22025619","citation_count":95,"is_preprint":false},{"pmid":"19729412","id":"PMC_19729412","title":"Genetic variation in GPR133 is associated with height: genome wide association study in the self-contained population of Sorbs.","date":"2009","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19729412","citation_count":61,"is_preprint":false},{"pmid":"27775701","id":"PMC_27775701","title":"GPR133 (ADGRD1), an adhesion G-protein-coupled receptor, is necessary for glioblastoma growth.","date":"2016","source":"Oncogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/27775701","citation_count":51,"is_preprint":false},{"pmid":"33623007","id":"PMC_33623007","title":"Control of oviductal fluid flow by the G-protein coupled receptor Adgrd1 is essential for murine embryo transit.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33623007","citation_count":40,"is_preprint":false},{"pmid":"34022221","id":"PMC_34022221","title":"Functional impact of intramolecular cleavage and dissociation of adhesion G protein-coupled receptor GPR133 (ADGRD1) on canonical signaling.","date":"2021","source":"The Journal of biological 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reports","url":"https://pubmed.ncbi.nlm.nih.gov/37354459","citation_count":15,"is_preprint":false},{"pmid":"27516204","id":"PMC_27516204","title":"Functional relevance of naturally occurring mutations in adhesion G protein-coupled receptor ADGRD1 (GPR133).","date":"2016","source":"BMC genomics","url":"https://pubmed.ncbi.nlm.nih.gov/27516204","citation_count":13,"is_preprint":false},{"pmid":"40583059","id":"PMC_40583059","title":"The mechanosensitive adhesion G protein-coupled receptor 133 (GPR133/ADGRD1) enhances bone formation.","date":"2025","source":"Signal transduction and targeted therapy","url":"https://pubmed.ncbi.nlm.nih.gov/40583059","citation_count":5,"is_preprint":false},{"pmid":"38758649","id":"PMC_38758649","title":"Modulation of GPR133 (ADGRD1) signaling by its intracellular interaction partner extended synaptotagmin 1.","date":"2024","source":"Cell 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Neither the N-terminal ectodomain nor cleavage at the GPCR proteolysis site is required for G protein signaling. Gs coupling was verified by Gαs siRNA knockdown, Gαs overexpression, chimeric Gq(s4) co-expression routing activity to PLC/IP pathway, and a transmembrane-domain missense mutation that abolished receptor activity without altering cell surface expression.\",\n      \"method\": \"siRNA knockdown of Gαs, overexpression of Gαs, chimeric G protein co-expression, missense mutagenesis, cAMP assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (knockdown, overexpression, chimeric protein, mutagenesis) in a single rigorous study, replicated across conditions\",\n      \"pmids\": [\"22025619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A short peptide sequence within the ectodomain of GPR133 (termed the Stachel sequence) functions as a tethered agonist; upon structural changes in the ectodomain, this intramolecular agonist is exposed to the seven-transmembrane domain to trigger G protein activation. The Stachel sequence shows high receptor specificity.\",\n      \"method\": \"Peptide agonist assay, zebrafish Stachel-mutant genetic rescue, exogenous peptide application in hypomorphic gpr126 mutants\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro peptide assay plus in vivo genetic rescue in zebrafish, replicated across two aGPCRs, independently confirmed by later structural work\",\n      \"pmids\": [\"25533341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"GPR133 knockdown reduces CD133+ glioblastoma stem cell prevalence, tumor cell proliferation, and tumorsphere formation in vitro, and markedly reduces tumor xenograft growth in vivo; the GPR133 knockdown phenotype is rescued by forskolin, indicating signaling is mediated through cAMP. GPR133 mRNA is transcriptionally upregulated by hypoxia in a HIF-1α-dependent manner.\",\n      \"method\": \"shRNA knockdown, tumorsphere assay, mouse xenograft implantation, forskolin rescue, HIF-1α-dependent transcription analysis\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function with defined cellular and in vivo phenotypes plus pharmacological rescue, replicated in patient-derived GBM cultures and xenograft model\",\n      \"pmids\": [\"27775701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Functional characterization of naturally occurring ADGRD1 missense variants identified several loss-of-function nsSNPs (A448D, Q600stop, C632fs, A761E, N795K) and one gain-of-function nsSNP (F383S) that significantly increased basal receptor activity.\",\n      \"method\": \"Site-directed mutagenesis, cAMP functional assay\",\n      \"journal\": \"BMC genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct mutagenesis and functional cAMP assay in a single lab study\",\n      \"pmids\": [\"27516204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"GPR133 undergoes autoproteolytic cleavage shortly after protein synthesis; the N-terminal fragment (NTF) and C-terminal fragment (CTF) remain noncovalently associated until the receptor is trafficked to the plasma membrane, where NTF-CTF dissociation occurs. Cleavage-competent WT GPR133 generates significantly more cAMP than the uncleavable H543R mutant. A PAR1-CTF/GPR133-NTF proxy system confirmed that thrombin-induced NTF shedding increases intracellular cAMP, supporting a model where NTF dissociation at the plasma membrane promotes receptor activation.\",\n      \"method\": \"Subcellular fractionation, co-immunoprecipitation, uncleavable point mutant (H543R), PAR1 chimeric proxy system with thrombin cleavage, cAMP assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal approaches (subcellular fractionation, Co-IP, mutagenesis, chimeric proxy system) in patient-derived GBM and HEK293T cells\",\n      \"pmids\": [\"34022221\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Adgrd1 is expressed on oviductal epithelium; female mice lacking Adgrd1 are sterile due to failure to relieve the ampullary-isthmic junction (AIJ) restraining mechanism, causing inappropriate retention of embryos in the oviduct. Post-ovulatory attenuation of tubal fluid flow is dysregulated in Adgrd1-deficient mice. The extracellular protein Plxdc2, displayed on cumulus cells, was identified as an activating ligand for Adgrd1 by a large-scale extracellular protein interaction screen.\",\n      \"method\": \"Constitutive knockout mice, oviductal fluid flow measurement, large-scale extracellular protein interaction screen (AVEXIS), embryo transit imaging\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — constitutive KO with specific reproductive phenotype, functional fluid-flow assay, and ligand identification by orthogonal protein interaction screen\",\n      \"pmids\": [\"33623007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM structures of ADGRD1 (and ADGRF1) in complex with Gs protein revealed that the stalk region preceding the first transmembrane helix acts as the tethered agonist by forming extensive interactions with the transmembrane domain; an autoproteolysis-deficient ADGRF1 structure showed a cleavage-independent manner of receptor activation. A conserved cascade of inter-helix interaction cores mediates stalk-induced activation.\",\n      \"method\": \"Cryo-EM structure determination, mutagenesis, functional signaling assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure with mutagenesis and functional validation, replicated across two aGPCRs in one rigorous study\",\n      \"pmids\": [\"35418679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Antibodies targeting the N-terminus of GPR133 increase cAMP in a concentration-dependent manner. This effect requires autoproteolytic cleavage: cells expressing the cleavage-deficient H543R mutant did not respond to antibody stimulation. Antibody treatment promotes release of the autoproteolytically cleaved NTF, supporting the model that NTF dissociation promotes receptor activation.\",\n      \"method\": \"Antibody treatment of HEK293T cells and patient-derived GBM cells, cAMP assay, cleavage-deficient mutant (H543R), immunoprecipitation of NTF from conditioned medium\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional antibody activation assay with cleavage-deficient mutant control and NTF detection, single lab\",\n      \"pmids\": [\"35447113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PTK7 is an extracellular binding partner of GPR133 in glioblastoma, identified by affinity proteomics. PTK7 binds the autoproteolytically generated NTF of GPR133 and its expression in trans increases GPR133 signaling. This allosteric effect requires GPR133 intramolecular cleavage and PTK7 anchoring in the plasma membrane. PTK7's allosteric action is additive with but topographically distinct from orthosteric Stachel peptide activation.\",\n      \"method\": \"Affinity proteomics, co-immunoprecipitation, cAMP assay, cleavage-deficient mutant, PTK7 transmembrane-anchoring requirement test, shRNA knockdown in GBM\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — affinity proteomics followed by reciprocal Co-IP, functional cAMP assay, mutagenesis/domain requirement experiments, and in vivo knockdown in GBM, all in a single thorough study\",\n      \"pmids\": [\"37354459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ESYT1, a Ca2+-dependent mediator of ER-plasma membrane bridge formation, is an intracellular interactor of GPR133 identified by proximity biotinylation proteomics. ESYT1 knockdown/knockout increases GPR133 signaling; overexpression suppresses it without altering plasma membrane GPR133 levels. The interaction requires the Ca2+-sensing C2C domain of ESYT1. Thapsigargin-mediated cytosolic Ca2+ elevation promotes ESYT1-GPR133 dissociation, relieving the signaling-suppressive effect.\",\n      \"method\": \"Proximity biotinylation proteomics (BioID), co-immunoprecipitation, ESYT1 KD/KO and overexpression, domain mutagenesis (C2C), thapsigargin treatment, cAMP assay, GBM tumor growth assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — proximity proteomics plus reciprocal Co-IP, domain-specific mutagenesis, pharmacological dissociation, multiple orthogonal functional readouts in single comprehensive study\",\n      \"pmids\": [\"38758649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Constitutive and osteoblast-specific knockouts of Gpr133/Adgrd1 in mice cause reduced cortical bone mass and trabecularization characteristic of osteoporosis, due to impaired osteoblast function and increased osteoclast activity. GPR133/ADGRD1 regulates osteoblast differentiation through a combined mechanism involving PTK7 interaction and mechanical forces (demonstrated by stretch assays in vitro and mechanical loading in vivo). Downstream signaling proceeds via cAMP-dependent activation of the β-catenin pathway. Pharmacological activation with agonist AP-970/43482503 (AP503) enhances osteoblast function and alleviates osteoporosis in ovariectomized mice.\",\n      \"method\": \"Constitutive and osteoblast-specific knockout mice, in vitro stretch assay, in vivo mechanical loading, cAMP assay, β-catenin pathway analysis, pharmacological agonist treatment, ovariectomy osteoporosis model\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — tissue-specific KO with defined skeletal phenotype, multiple in vitro and in vivo mechanistic experiments including mechanosensing and pathway analysis\",\n      \"pmids\": [\"40583059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The small molecule GL64, identified as a selective ADGRD1 agonist, activates ADGRD1 by mimicking the Stachel sequence. ADGRD1 negatively regulates osteoclastogenesis via the cAMP-PKA-NFATC1 signaling pathway, and GL64 administration prevents bone loss in an ovariectomy mouse model.\",\n      \"method\": \"Small-molecule agonist identification, cAMP assay, PKA and NFATC1 pathway analysis, osteoclast differentiation assay, ovariectomy mouse model\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — small-molecule tool compound with defined mechanism of action (Stachel mimicry), downstream pathway dissection, and in vivo validation\",\n      \"pmids\": [\"40644539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GPR133 exhibits constitutive self-activation via its Stachel sequence and can activate downstream G13 signaling in addition to Gs. A cryo-EM structure of the GPR133-GAIN-miniGα13 complex was resolved at 3.51 Å, revealing both conserved and distinct features compared to the previously resolved GPR133-CTF-Gs complex.\",\n      \"method\": \"Cryo-EM structure determination (3.51 Å), in vitro reconstitution of GPR133-GAIN-miniGα13 complex, G13 signaling assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — high-quality cryo-EM structure with in vitro reconstitution, but single lab and newly reported finding without independent replication\",\n      \"pmids\": [\"40570642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GPR133 upregulation in decidual macrophages (caused by promoter hypomethylation) impairs phagocytic function; GPR133 knockdown in THP-1 macrophages significantly improves phagocytic function.\",\n      \"method\": \"5-Aza-dC demethylation, siRNA/shRNA knockdown, phagocytosis assay in decidual macrophages and THP-1 cells\",\n      \"journal\": \"Epigenetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — functional knockdown with defined cellular phenotype (phagocytosis assay), supported by epigenetic manipulation, single lab\",\n      \"pmids\": [\"38564758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ADGRD1 promotes differentiation of adipose progenitor cells (APCs) in vitro and in vivo. In an obese mouse model (high-fat diet), gain-of-function and loss-of-function studies validated that ADGRD1 promotes adipogenesis and improves metabolic homeostasis. Transcription factors MEF2D and TCF12 were identified as regulators of ADGRD1 expression.\",\n      \"method\": \"Single-nucleus sequencing trajectory inference, primary APC differentiation assay, gain- and loss-of-function in HFD mouse model, ChIP-seq and RNA-seq analysis\",\n      \"journal\": \"Science China. Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — in vitro and in vivo gain/loss-of-function with metabolic phenotype, supported by multi-omic transcription factor analysis, single lab\",\n      \"pmids\": [\"39821834\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ADGRD1/GPR133 is an adhesion GPCR that is autoproteolytically cleaved at its GAIN domain GPS site shortly after synthesis; the NTF and CTF remain noncovalently associated until the receptor reaches the plasma membrane, where NTF dissociation exposes the Stachel tethered-agonist sequence in the stalk region, which inserts into the transmembrane domain to activate primarily Gs (elevating cAMP) and also G13 signaling. Cryo-EM structures have resolved the molecular basis of this stalk–TMD interaction. Receptor activity is positively modulated by the extracellular binding partner PTK7 (which acts allosterically on the NTF) and by mechanical forces, while it is negatively regulated intracellularly by ESYT1 in a Ca2+-dependent manner. Physiologically, ADGRD1 controls oviductal fluid flow and embryo transit, promotes osteoblast differentiation and bone formation via cAMP/β-catenin signaling, inhibits osteoclastogenesis via cAMP-PKA-NFATC1, promotes adipogenesis, and supports glioblastoma stem-cell growth.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ADGRD1 (GPR133) is an adhesion G protein-coupled receptor that transduces extracellular and mechanical cues into intracellular cAMP signaling to control reproductive, skeletal, metabolic, and tumor-cell biology [#0, #5, #10]. It couples to Gs to activate adenylyl cyclase and elevate cAMP, and also engages G13 [#0, #12]. Activation is governed by a tethered-agonist mechanism: the receptor undergoes autoproteolytic cleavage at its GAIN/GPS site shortly after synthesis, generating an N-terminal fragment (NTF) that stays noncovalently associated with the C-terminal fragment until it dissociates at the plasma membrane, exposing the intramolecular Stachel sequence that inserts into the transmembrane domain to drive signaling [#1, #4, #6]. Cryo-EM structures have resolved how the stalk engages the transmembrane domain through a conserved cascade of inter-helix interaction cores, including a cleavage-independent mode of activation [#6, #12]. Receptor output is tuned by binding partners: the extracellular protein PTK7 binds the NTF and allosterically potentiates signaling in a cleavage-dependent, membrane-anchored manner topographically distinct from Stachel activation [#8], mechanical force enhances activity [#10], and the Ca2+-dependent ER-plasma membrane tether ESYT1 binds intracellularly to suppress signaling, an inhibition relieved by cytosolic Ca2+ elevation [#9]. Plxdc2 displayed on cumulus cells acts as an activating ligand controlling oviductal fluid flow and embryo transit, loss of which renders female mice sterile [#5]. Through cAMP signaling, ADGRD1 promotes osteoblast differentiation and bone formation via the \\u03b2-catenin pathway and inhibits osteoclastogenesis via cAMP-PKA-NFATC1, with its loss producing osteoporosis-like skeletal defects [#10, #11]; it also promotes adipogenesis and metabolic homeostasis [#14] and supports glioblastoma stem-cell growth, where it is upregulated by HIF-1\\u03b1-dependent hypoxic transcription [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established the core transduction logic of the receptor by showing GPR133 couples to Gs and elevates cAMP independently of its ectodomain or proteolytic cleavage.\",\n      \"evidence\": \"G\\u03b1s knockdown/overexpression, chimeric G protein routing, missense mutagenesis and cAMP assay\",\n      \"pmids\": [\"22025619\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the physiological activating input\", \"Mechanism of receptor activation at the molecular level unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the activation mechanism by identifying the Stachel sequence as an intramolecular tethered agonist that engages the seven-transmembrane domain.\",\n      \"evidence\": \"Peptide agonist assay plus zebrafish Stachel-mutant genetic rescue across two aGPCRs\",\n      \"pmids\": [\"25533341\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ectodomain conformational change exposes the Stachel in vivo not resolved\", \"Endogenous triggers of exposure unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Linked GPR133 to a disease context by showing it sustains glioblastoma stem-cell growth through cAMP and is transcriptionally induced by hypoxia.\",\n      \"evidence\": \"shRNA knockdown, tumorsphere and xenograft assays, forskolin rescue, HIF-1\\u03b1-dependent transcription analysis\",\n      \"pmids\": [\"27775701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous activating ligand in GBM not identified\", \"Downstream effectors beyond cAMP not mapped\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connected receptor function to natural sequence variation by functionally classifying loss- and gain-of-function ADGRD1 missense variants.\",\n      \"evidence\": \"Site-directed mutagenesis and cAMP functional assay\",\n      \"pmids\": [\"27516204\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab functional characterization without in vivo validation\", \"Physiological consequences of variants unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved how cleavage controls activation by showing NTF-CTF dissociation at the plasma membrane promotes signaling, with cleavage-competent receptor producing more cAMP than the uncleavable mutant.\",\n      \"evidence\": \"Subcellular fractionation, Co-IP, H543R uncleavable mutant, PAR1-CTF chimeric proxy with thrombin, cAMP assay\",\n      \"pmids\": [\"34022221\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological driver of NTF dissociation in native tissue unknown\", \"Whether dissociation is required or sufficient for full activation not fully resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined a physiological role and an activating ligand by showing Adgrd1 controls oviductal fluid flow and embryo transit, with Plxdc2 on cumulus cells acting as ligand.\",\n      \"evidence\": \"Constitutive KO mice, oviductal fluid-flow measurement, AVEXIS interaction screen, embryo transit imaging\",\n      \"pmids\": [\"33623007\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signaling pathway downstream of Plxdc2-Adgrd1 in oviduct not dissected\", \"Whether Plxdc2 acts via the Stachel mechanism unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided the structural basis of tethered-agonist activation by resolving cryo-EM structures showing the stalk engages the transmembrane domain through a conserved inter-helix cascade, including cleavage-independent activation.\",\n      \"evidence\": \"Cryo-EM of ADGRD1/ADGRF1-Gs complexes, mutagenesis, functional signaling assays\",\n      \"pmids\": [\"35418679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structures captured Gs-bound state only\", \"Conformational transition from inactive to active state not visualized\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated that NTF-directed antibodies can pharmacologically activate the receptor in a cleavage-dependent manner, validating NTF dissociation as a druggable activation step.\",\n      \"evidence\": \"Antibody treatment of HEK293T and GBM cells, cAMP assay, H543R control, NTF immunoprecipitation\",\n      \"pmids\": [\"35447113\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study without independent replication\", \"Therapeutic applicability in vivo not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified PTK7 as an extracellular allosteric positive modulator that binds the NTF and potentiates signaling distinctly from orthosteric Stachel activation.\",\n      \"evidence\": \"Affinity proteomics, reciprocal Co-IP, cAMP assay, cleavage-deficient mutant, PTK7 membrane-anchoring test, GBM knockdown\",\n      \"pmids\": [\"37354459\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of PTK7-NTF interaction unresolved\", \"Whether PTK7 modulation operates in non-GBM tissues not addressed here\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed intracellular negative regulation by showing ESYT1 binds GPR133 in a Ca2+-dependent manner to suppress signaling, an inhibition relieved by cytosolic Ca2+ elevation.\",\n      \"evidence\": \"BioID proximity proteomics, reciprocal Co-IP, ESYT1 KD/KO/overexpression, C2C domain mutagenesis, thapsigargin, cAMP and GBM growth assays\",\n      \"pmids\": [\"38758649\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ESYT1 binding mechanically suppresses receptor signaling unclear\", \"Physiological Ca2+ stimuli that gate this regulation in vivo unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended receptor function to innate immunity by showing GPR133 upregulation impairs macrophage phagocytosis.\",\n      \"evidence\": \"5-Aza-dC demethylation, siRNA/shRNA knockdown, phagocytosis assays in decidual and THP-1 macrophages\",\n      \"pmids\": [\"38564758\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab functional study\", \"Signaling pathway linking GPR133 to phagocytic suppression not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established a skeletal role by showing GPR133 promotes osteoblast differentiation and bone formation via mechanical force, PTK7, and cAMP/\\u03b2-catenin signaling, with a tractable agonist alleviating osteoporosis.\",\n      \"evidence\": \"Constitutive and osteoblast-specific KO mice, stretch and mechanical loading assays, \\u03b2-catenin pathway analysis, AP503 agonist in ovariectomy model\",\n      \"pmids\": [\"40583059\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link between mechanical force and Stachel/NTF dynamics not resolved\", \"How cAMP feeds into \\u03b2-catenin mechanistically not fully defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed ADGRD1 negatively regulates osteoclastogenesis via cAMP-PKA-NFATC1 and identified a Stachel-mimicking small-molecule agonist that prevents bone loss.\",\n      \"evidence\": \"GL64 small-molecule agonist, cAMP/PKA/NFATC1 pathway analysis, osteoclast differentiation assay, ovariectomy model\",\n      \"pmids\": [\"40644539\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-type specificity of GL64 action not fully delineated\", \"Whether osteoblast and osteoclast effects are cell-autonomous not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanded signaling repertoire by demonstrating G13 coupling and constitutive Stachel-driven self-activation, with a cryo-EM GAIN-miniG\\u03b113 structure.\",\n      \"evidence\": \"Cryo-EM at 3.51 \\u00c5, in vitro reconstitution of GPR133-GAIN-miniG\\u03b113, G13 signaling assay\",\n      \"pmids\": [\"40570642\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab structure without independent replication\", \"Physiological contexts requiring G13 versus Gs signaling unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated a metabolic role by showing ADGRD1 promotes adipocyte progenitor differentiation and improves metabolic homeostasis, with MEF2D and TCF12 controlling its expression.\",\n      \"evidence\": \"Single-nucleus trajectory inference, primary APC differentiation, gain/loss-of-function in HFD mice, ChIP-seq and RNA-seq\",\n      \"pmids\": [\"39821834\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Downstream signaling in adipocytes not connected to known cAMP mechanism\", \"Activating ligand in adipose tissue not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The endogenous activating inputs (ligands versus mechanical force) and the conformational sequence that exposes the Stachel in native tissues remain incompletely defined across the receptor's diverse physiological roles.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model of how tissue-specific ligands, force, and partner proteins converge on Stachel exposure\", \"Inactive-state structure not resolved\", \"Tissue-specific Gs versus G13 bias not mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 6, 12]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4, 5, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 6, 8, 9]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [5, 10, 14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PTK7\", \"ESYT1\", \"PLXDC2\", \"GNAS\", \"GNA13\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":7,"faith_total":7,"faith_pct":100.0}}