{"gene":"ITGB3BP","run_date":"2026-06-10T01:55:23","timeline":{"discoveries":[{"year":1999,"finding":"NRIF3 (ITGB3BP) localizes to the cell nucleus and acts as a coactivator that specifically interacts with thyroid hormone receptor (TR) and retinoid X receptor (RXR) in a ligand-dependent fashion, but not with retinoic acid receptor, vitamin D receptor, progesterone receptor, glucocorticoid receptor, or estrogen receptor. A novel C-terminal LXXIL module (RID1) mediates the receptor interaction, while an N-terminal LXXLL motif (RID2) plays a minor modulatory role.","method":"Yeast two-hybrid, in vitro binding assays, fluorescence microscopy, domain deletion and mutagenesis analyses, cotransfection transactivation assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (Y2H, in vitro binding, mutagenesis, functional transactivation assays), receptor specificity rigorously mapped","pmids":["10490654"],"is_preprint":false},{"year":2001,"finding":"NRIF3 employs a bivalent interaction model using both C-terminal RID1 (LXXIL) and N-terminal RID2 (LXXLL) modules to cooperatively interact with TR or RXR dimers; spacing between modules is important for affinity. A dimerization domain was mapped to residues 84–112 (predicted coiled-coil/leucine zipper). An autonomous transactivation domain (AD1) was mapped to the C-terminus, and a repression domain (RepD1) was mapped to residues 20–50 at the N-terminal portion. A single amino acid change Ser28Ala in RepD1 abolished transrepression, suggesting phosphorylation at this site regulates coregulatory function.","method":"Deletion and mutagenesis analyses, Gal4 fusion reporter assays, cotransfection assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — systematic mutagenesis and deletion mapping with multiple functional reporters in one rigorous study","pmids":["11713274"],"is_preprint":false},{"year":1999,"finding":"TAP20 (ITGB3BP) is a PKCθ-dependent protein in endothelial cells that physically interacts with the β5 integrin cytoplasmic domain. Overexpression of TAP20 decreased cell adhesion, enhanced migration on vitronectin, and promoted tube formation in 3D culture; these effects were prevented by an anti-integrin αvβ5 antibody. TAP20 also decreased focal adhesion formation in αvβ3-deficient cells.","method":"Protein coprecipitation, immunoblotting, overexpression with functional adhesion, migration, and tube formation assays, antibody blocking experiments","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal coprecipitation plus multiple orthogonal functional assays (adhesion, migration, tube formation, antibody blocking) in one study","pmids":["10579726"],"is_preprint":false},{"year":2004,"finding":"NRIF3 (ITGB3BP) interacts with metastasis-associated protein 1 (MTA1) both in vitro and in vivo; NRIF3 binds to the C-terminal region of MTA1 via its N-terminal LXXLL-containing region. NRIF3 functions as an ER coactivator, associates with endogenous ERα and its target gene promoter chromatin, and MTA1 represses NRIF3-mediated ERE-driven transcription by interfering with NRIF3's chromatin association.","method":"Yeast two-hybrid screen, in vitro binding assay, co-immunoprecipitation (in vivo), chromatin immunoprecipitation (ChIP), transactivation reporter assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal in vitro and in vivo binding, ChIP, and functional reporter assays in one study","pmids":["15254226"],"is_preprint":false},{"year":2004,"finding":"NRIF3 (ITGB3BP) and its family members induce rapid apoptosis in breast cancer cells via a novel death domain (DD1) mapped to a 30-amino-acid region. DD1-induced apoptosis proceeds through a caspase 2-mediated pathway involving mitochondrial membrane permeabilization but does not require other caspases. Cytotoxicity is cell type specific, selectively killing breast cancer cells.","method":"Expression of NRIF3/DD1 in cell lines, domain deletion/mutagenesis, caspase inhibitor studies, mitochondrial membrane permeability assays, cell viability assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain mapping, specific caspase pathway dissection with pharmacological inhibitors, multiple orthogonal assays","pmids":["15082778"],"is_preprint":false},{"year":2008,"finding":"Pak1 phosphorylates NRIF3 (ITGB3BP) at Serine 28 in vitro and in vivo. Phosphorylation of Ser28 increases NRIF3's co-activator activity, its interaction with ERα, its nuclear localization, and its recruitment to endogenous ERα target gene promoters. A phospho-mimicking mutant (Ser28Glu) phenocopied activated Pak1 in enhancing ERα transactivation and estrogen responsiveness.","method":"In vitro kinase assay, co-immunoprecipitation, phospho-mimicking and non-phosphorylatable mutagenesis, transactivation reporter assays, ChIP, subcellular localization imaging","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro kinase assay combined with mutagenesis, ChIP, and functional transactivation assays; multiple orthogonal methods","pmids":["18521086"],"is_preprint":false},{"year":2022,"finding":"CENP-R (ITGB3BP) synergizes with CENP-OPQU to regulate kinetochore-microtubule attachment stability and accurate chromosome segregation in mitosis. Aurora B-mediated phosphorylation of CENP-R weakens its kinetochore localization and disrupts its binding with CENP-U, thereby promoting correction of improper kinetochore-microtubule attachments.","method":"Phospho-mimicking mutagenesis, kinetochore localization assays, co-immunoprecipitation (CENP-R/CENP-U interaction), chromosome segregation assays, mitosis imaging","journal":"Journal of molecular cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — mutagenesis, co-IP, localization, and functional chromosome segregation readouts combined in one rigorous study","pmids":["36069839"],"is_preprint":false},{"year":2017,"finding":"CENP-R (ITGB3BP) is a constitutive kinetochore component throughout the cell cycle in vertebrates and is part of the CENP-O complex (CENP-O/P/Q/R/U). CENP-R-deficient (Cenp-r−/−) mice are viable, indicating it is not essential for normal development, but loss of CENP-R leads to increased early tumor formation and altered apoptosis/proliferation balance in papillomas, suggesting a context-dependent role in cancer progression.","method":"Knockout mouse model (Cenp-r−/−), DMBA/TPA chemical carcinogenesis protocol, histological analysis of proliferation and apoptosis markers","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockout mouse with defined tumor biology readout, but mechanism is inferred from in vivo phenotype without direct biochemical dissection","pmids":["28795467"],"is_preprint":false},{"year":2025,"finding":"CENP-R (ITGB3BP) interacts with EB1 via CENP-R's N-terminal intrinsic disorder region and EB1's end-binding homology domain. EB1 and CENP-R undergo co-condensation (phase separation). An EB1-binding-defective CENP-R mutant perturbs chromosome oscillations, indicating that EB1–CENP-R condensation forms a physical link between the inner kinetochore and dynamic spindle microtubule plus-ends to drive chromosome oscillations in mitosis.","method":"NMR interaction mapping, biochemical co-condensation assays, EB1-binding-defective CENP-R mutant expression, live-cell chromosome oscillation imaging","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — NMR structural mapping combined with biochemical condensation assays and functional mutant imaging across orthogonal methods","pmids":["40349345"],"is_preprint":false},{"year":2025,"finding":"ITGB3BP protein is subject to ubiquitin-mediated degradation promoted by its interaction with MDM2, as induced by lncRNA NR_045147. ITGB3BP upregulation promotes osteogenic differentiation and migration of periodontal ligament stem cells and enhances mitochondrial respiration.","method":"Ubiquitination assay, western blotting for protein stability, osteogenic differentiation assays, migration assays, Seahorse XF mitochondrial respiration analysis, in vivo calvarial defect model","journal":"Stem cells translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination assays with functional readouts, single lab, mechanism linked to MDM2 interaction but indirect (via lncRNA context)","pmids":["39674578"],"is_preprint":false}],"current_model":"ITGB3BP (NRIF3/TAP20/CENP-R) is a multifunctional protein: as a nuclear receptor coregulator it uses bivalent LXXIL/LXXLL motifs to specifically co-activate TR and RXR (and ERα following Pak1-mediated Ser28 phosphorylation), while its N-terminal RepD1 can repress transcription; it also contains a death domain (DD1) that triggers caspase 2-dependent, mitochondria-involved apoptosis selectively in breast cancer cells; as a structural kinetochore component of the CENP-O complex it is phosphorylated by Aurora B to disrupt CENP-U binding and correct improper kinetochore-microtubule attachments, and co-condenses with EB1 to link the inner kinetochore to dynamic microtubule plus-ends for chromosome oscillations; additionally, it interacts with the β5 integrin cytoplasmic domain downstream of PKCθ to modulate endothelial cell adhesion and migration."},"narrative":{"mechanistic_narrative":"ITGB3BP (NRIF3/TAP20/CENP-R) is a multifunctional protein operating in two largely distinct arenas: nuclear receptor coregulation and mitotic kinetochore function [PMID:10490654, PMID:36069839]. As a nuclear coactivator it localizes to the nucleus and uses a bivalent interaction mechanism—a C-terminal LXXIL module (RID1) as the primary receptor-contact and an N-terminal LXXLL motif (RID2) as a modulatory contact—to engage thyroid hormone receptor and RXR dimers in a ligand-dependent manner, with inter-module spacing governing affinity [PMID:10490654, PMID:11713274]. Its C-terminus harbors an autonomous transactivation domain while an N-terminal RepD1 (residues 20–50) confers transrepression that is abolished by Ser28Ala, defining this residue as a regulatory phospho-site [PMID:11713274]. Pak1 phosphorylates Ser28, enhancing nuclear localization, ERα binding, recruitment to ERα target promoters, and estrogen responsiveness, thereby extending coactivator function to ERα [PMID:18521086]; MTA1 binds the N-terminal LXXLL region and represses NRIF3-driven ERE transcription by blocking its chromatin association [PMID:15254226]. A separate 30-amino-acid death domain (DD1) drives caspase 2-dependent, mitochondria-involving apoptosis selectively in breast cancer cells [PMID:15082778]. In mitosis, ITGB3BP is a constitutive kinetochore component of the CENP-O complex (CENP-O/P/Q/R/U) that regulates kinetochore–microtubule attachment stability; Aurora B phosphorylation weakens its kinetochore localization and disrupts its binding to CENP-U to correct erroneous attachments [PMID:36069839, PMID:28795467], and co-condensation with EB1 via its N-terminal disordered region links the inner kinetochore to dynamic microtubule plus-ends to drive chromosome oscillations [PMID:40349345]. Independently, TAP20 acts downstream of PKCθ in endothelial cells, binding the β5 integrin cytoplasmic domain to reduce adhesion and promote migration and tube formation [PMID:10579726], and its protein levels are controlled by MDM2-promoted ubiquitin-mediated degradation [PMID:39674578].","teleology":[{"year":1999,"claim":"Established ITGB3BP as a nuclear receptor coactivator with specificity, answering whether it acts broadly or selectively across receptors.","evidence":"Yeast two-hybrid, in vitro binding, mutagenesis and transactivation assays mapping a C-terminal LXXIL (RID1) module","pmids":["10490654"],"confidence":"High","gaps":["Did not resolve stoichiometry of receptor-dimer engagement","No structural model of RID1 binding","Endogenous target genes not defined"]},{"year":1999,"claim":"Defined a separate cytoplasmic identity for the same protein, showing it links PKCθ signaling to integrin-dependent endothelial behavior.","evidence":"Coprecipitation with β5 integrin cytoplasmic domain plus adhesion, migration, tube-formation and antibody-blocking assays","pmids":["10579726"],"confidence":"High","gaps":["Molecular basis of integrin-tail binding not mapped","Relationship to the nuclear coactivator role unexplained","Downstream signaling effectors unidentified"]},{"year":2001,"claim":"Resolved how the coregulator engages receptors and how its activity is partitioned into activating and repressing domains, defining Ser28 as a regulatory node.","evidence":"Deletion/mutagenesis mapping of bivalent RID modules, dimerization domain, AD1, and RepD1 with Gal4 reporter assays","pmids":["11713274"],"confidence":"High","gaps":["Kinase responsible for Ser28 phosphorylation not yet identified","Physiological context of repression versus activation unclear"]},{"year":2004,"claim":"Revealed a coregulatory antagonism, showing MTA1 represses ITGB3BP-driven ER signaling by displacing it from chromatin.","evidence":"Y2H, in vitro and in vivo binding, ChIP, and ERE reporter assays","pmids":["15254226"],"confidence":"High","gaps":["Mechanism of chromatin displacement not structurally defined","Generality across other receptor target genes untested"]},{"year":2004,"claim":"Identified a pro-apoptotic activity distinct from transcription, defining a death domain that selectively kills breast cancer cells.","evidence":"Domain mapping, caspase inhibitor studies, and mitochondrial permeability assays in cell lines","pmids":["15082778"],"confidence":"High","gaps":["Molecular basis of cell-type selectivity unknown","Upstream trigger of DD1-mediated apoptosis unidentified","Endogenous relevance versus overexpression unclear"]},{"year":2008,"claim":"Connected upstream signaling to coregulator output, showing Pak1 phosphorylation of Ser28 activates ITGB3BP toward ERα.","evidence":"In vitro kinase assay, phospho-mimic/non-phosphorylatable mutants, ChIP, and transactivation assays","pmids":["18521086"],"confidence":"High","gaps":["Interplay between Ser28 activation here and RepD1 repression unresolved","Other phospho-regulated receptors not surveyed"]},{"year":2017,"claim":"Placed ITGB3BP in the constitutive kinetochore CENP-O complex and tested organismal requirement, showing it is dispensable for development but modifies tumor formation.","evidence":"Cenp-r−/− knockout mouse with DMBA/TPA carcinogenesis and histological proliferation/apoptosis analysis","pmids":["28795467"],"confidence":"Medium","gaps":["Tumor phenotype mechanism inferred from in vivo readout without biochemical dissection","Link between kinetochore role and tumorigenesis not defined"]},{"year":2022,"claim":"Defined how the kinetochore role is regulated, showing Aurora B phosphorylation disengages CENP-R from CENP-U to correct erroneous attachments.","evidence":"Phospho-mimic mutagenesis, kinetochore localization, CENP-R/CENP-U co-IP, and chromosome segregation assays","pmids":["36069839"],"confidence":"High","gaps":["Structural basis of CENP-R/CENP-U interface not solved","Aurora B phospho-sites not enumerated here"]},{"year":2025,"claim":"Established a physical mechanism coupling the inner kinetochore to microtubule plus-ends, showing CENP-R co-condenses with EB1 to drive chromosome oscillations.","evidence":"NMR interaction mapping, co-condensation assays, and EB1-binding-defective mutant with live-cell oscillation imaging","pmids":["40349345"],"confidence":"High","gaps":["Regulation of the condensation in vivo not defined","Interplay with Aurora B-controlled CENP-U binding unexplored"]},{"year":2025,"claim":"Identified post-translational control of ITGB3BP abundance and a metabolic/differentiation role in stem cells.","evidence":"Ubiquitination and stability assays, MDM2 interaction, osteogenic and migration assays, Seahorse respiration, and calvarial defect model","pmids":["39674578"],"confidence":"Medium","gaps":["MDM2-mediated degradation linked indirectly via lncRNA context","Single lab, mechanism of mitochondrial enhancement undefined"]},{"year":null,"claim":"How the protein's nuclear coregulator, apoptotic, kinetochore, and integrin-signaling roles are coordinated within one cell, and whether they share a common regulatory logic, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model linking the distinct functional modules","Isoform/context determinants of which role is active are unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1,3,5]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,3,5]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[6,8]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[8]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,5]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[6,7]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,1,3,5]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6,7,8]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4]}],"complexes":["CENP-O complex (CENP-O/P/Q/R/U)","kinetochore"],"partners":["THRA","RXRA","ESR1","MTA1","PAK1","ITGB5","CENP-U","EB1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13352","full_name":"Centromere protein R","aliases":["Beta-3-endonexin","Integrin beta-3-binding protein","Nuclear receptor-interacting factor 3"],"length_aa":177,"mass_kda":20.2,"function":"Transcription coregulator that can have both coactivator and corepressor functions. Isoform 1, but not other isoforms, is involved in the coactivation of nuclear receptors for retinoid X (RXRs) and thyroid hormone (TRs) in a ligand-dependent fashion. In contrast, it does not coactivate nuclear receptors for retinoic acid, vitamin D, progesterone receptor, nor glucocorticoid. Acts as a coactivator for estrogen receptor alpha. Acts as a transcriptional corepressor via its interaction with the NFKB1 NF-kappa-B subunit, possibly by interfering with the transactivation domain of NFKB1. Induces apoptosis in breast cancer cells, but not in other cancer cells, via a caspase-2 mediated pathway that involves mitochondrial membrane permeabilization but does not require other caspases. May also act as an inhibitor of cyclin A-associated kinase. Also acts a component of the CENPA-CAD (nucleosome distal) complex, a complex recruited to centromeres which is involved in assembly of kinetochore proteins, mitotic progression and chromosome segregation. May be involved in incorporation of newly synthesized CENPA into centromeres via its interaction with the CENPA-NAC complex","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q13352/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ITGB3BP","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ITGB3BP","total_profiled":1310},"omim":[{"mim_id":"605494","title":"INTEGRIN, BETA-3, BINDING PROTEIN OF; ITGB3BP","url":"https://www.omim.org/entry/605494"},{"mim_id":"164750","title":"OMPHALOCELE, AUTOSOMAL","url":"https://www.omim.org/entry/164750"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ITGB3BP"},"hgnc":{"alias_symbol":["NRIF3","HSU37139","TAP20","CENPR"],"prev_symbol":[]},"alphafold":{"accession":"Q13352","domains":[{"cath_id":"1.10.287","chopping":"83-155","consensus_level":"high","plddt":83.4721,"start":83,"end":155}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13352","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13352-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13352-F1-predicted_aligned_error_v6.png","plddt_mean":72.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ITGB3BP","jax_strain_url":"https://www.jax.org/strain/search?query=ITGB3BP"},"sequence":{"accession":"Q13352","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13352.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13352/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13352"}},"corpus_meta":[{"pmid":"10490654","id":"PMC_10490654","title":"NRIF3 is a novel coactivator mediating functional specificity of nuclear hormone receptors.","date":"1999","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/10490654","citation_count":51,"is_preprint":false},{"pmid":"15254226","id":"PMC_15254226","title":"Metastasis-associated protein 1 interacts with NRIF3, an estrogen-inducible nuclear receptor coregulator.","date":"2004","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15254226","citation_count":36,"is_preprint":false},{"pmid":"10579726","id":"PMC_10579726","title":"Enhancement of endothelial cell migration and in vitro tube formation by TAP20, a novel beta 5 integrin-modulating, PKC theta-dependent protein.","date":"1999","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/10579726","citation_count":35,"is_preprint":false},{"pmid":"15082778","id":"PMC_15082778","title":"The NRIF3 family of transcriptional coregulators induces rapid and profound apoptosis in breast cancer cells.","date":"2004","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15082778","citation_count":27,"is_preprint":false},{"pmid":"11713274","id":"PMC_11713274","title":"Domain structure of the NRIF3 family of coregulators suggests potential dual roles in transcriptional regulation.","date":"2001","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/11713274","citation_count":26,"is_preprint":false},{"pmid":"18521086","id":"PMC_18521086","title":"Serine 28 phosphorylation of NRIF3 confers its co-activator function for estrogen receptor-alpha transactivation.","date":"2008","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/18521086","citation_count":10,"is_preprint":false},{"pmid":"28795467","id":"PMC_28795467","title":"CENP-R acts bilaterally as a tumor suppressor and as an oncogene in the two-stage skin carcinogenesis model.","date":"2017","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/28795467","citation_count":9,"is_preprint":false},{"pmid":"36069839","id":"PMC_36069839","title":"Phosphorylation of CENP-R by Aurora B regulates kinetochore-microtubule attachment for accurate chromosome segregation.","date":"2022","source":"Journal of molecular cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/36069839","citation_count":8,"is_preprint":false},{"pmid":"39674578","id":"PMC_39674578","title":"LncRNA NR_045147 modulates osteogenic differentiation and migration in PDLSCs via ITGB3BP degradation and mitochondrial dysfunction.","date":"2025","source":"Stem cells translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39674578","citation_count":6,"is_preprint":false},{"pmid":"40349345","id":"PMC_40349345","title":"Condensation-dependent multivalent interactions of EB1 and CENP-R regulate chromosome oscillations in mitosis.","date":"2025","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/40349345","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7434,"output_tokens":3034,"usd":0.033906,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10440,"output_tokens":3595,"usd":0.071038,"stage2_stop_reason":"end_turn"},"total_usd":0.104944,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"NRIF3 (ITGB3BP) localizes to the cell nucleus and acts as a coactivator that specifically interacts with thyroid hormone receptor (TR) and retinoid X receptor (RXR) in a ligand-dependent fashion, but not with retinoic acid receptor, vitamin D receptor, progesterone receptor, glucocorticoid receptor, or estrogen receptor. A novel C-terminal LXXIL module (RID1) mediates the receptor interaction, while an N-terminal LXXLL motif (RID2) plays a minor modulatory role.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding assays, fluorescence microscopy, domain deletion and mutagenesis analyses, cotransfection transactivation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (Y2H, in vitro binding, mutagenesis, functional transactivation assays), receptor specificity rigorously mapped\",\n      \"pmids\": [\"10490654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"NRIF3 employs a bivalent interaction model using both C-terminal RID1 (LXXIL) and N-terminal RID2 (LXXLL) modules to cooperatively interact with TR or RXR dimers; spacing between modules is important for affinity. A dimerization domain was mapped to residues 84–112 (predicted coiled-coil/leucine zipper). An autonomous transactivation domain (AD1) was mapped to the C-terminus, and a repression domain (RepD1) was mapped to residues 20–50 at the N-terminal portion. A single amino acid change Ser28Ala in RepD1 abolished transrepression, suggesting phosphorylation at this site regulates coregulatory function.\",\n      \"method\": \"Deletion and mutagenesis analyses, Gal4 fusion reporter assays, cotransfection assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — systematic mutagenesis and deletion mapping with multiple functional reporters in one rigorous study\",\n      \"pmids\": [\"11713274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"TAP20 (ITGB3BP) is a PKCθ-dependent protein in endothelial cells that physically interacts with the β5 integrin cytoplasmic domain. Overexpression of TAP20 decreased cell adhesion, enhanced migration on vitronectin, and promoted tube formation in 3D culture; these effects were prevented by an anti-integrin αvβ5 antibody. TAP20 also decreased focal adhesion formation in αvβ3-deficient cells.\",\n      \"method\": \"Protein coprecipitation, immunoblotting, overexpression with functional adhesion, migration, and tube formation assays, antibody blocking experiments\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal coprecipitation plus multiple orthogonal functional assays (adhesion, migration, tube formation, antibody blocking) in one study\",\n      \"pmids\": [\"10579726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"NRIF3 (ITGB3BP) interacts with metastasis-associated protein 1 (MTA1) both in vitro and in vivo; NRIF3 binds to the C-terminal region of MTA1 via its N-terminal LXXLL-containing region. NRIF3 functions as an ER coactivator, associates with endogenous ERα and its target gene promoter chromatin, and MTA1 represses NRIF3-mediated ERE-driven transcription by interfering with NRIF3's chromatin association.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro binding assay, co-immunoprecipitation (in vivo), chromatin immunoprecipitation (ChIP), transactivation reporter assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal in vitro and in vivo binding, ChIP, and functional reporter assays in one study\",\n      \"pmids\": [\"15254226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"NRIF3 (ITGB3BP) and its family members induce rapid apoptosis in breast cancer cells via a novel death domain (DD1) mapped to a 30-amino-acid region. DD1-induced apoptosis proceeds through a caspase 2-mediated pathway involving mitochondrial membrane permeabilization but does not require other caspases. Cytotoxicity is cell type specific, selectively killing breast cancer cells.\",\n      \"method\": \"Expression of NRIF3/DD1 in cell lines, domain deletion/mutagenesis, caspase inhibitor studies, mitochondrial membrane permeability assays, cell viability assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain mapping, specific caspase pathway dissection with pharmacological inhibitors, multiple orthogonal assays\",\n      \"pmids\": [\"15082778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Pak1 phosphorylates NRIF3 (ITGB3BP) at Serine 28 in vitro and in vivo. Phosphorylation of Ser28 increases NRIF3's co-activator activity, its interaction with ERα, its nuclear localization, and its recruitment to endogenous ERα target gene promoters. A phospho-mimicking mutant (Ser28Glu) phenocopied activated Pak1 in enhancing ERα transactivation and estrogen responsiveness.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, phospho-mimicking and non-phosphorylatable mutagenesis, transactivation reporter assays, ChIP, subcellular localization imaging\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro kinase assay combined with mutagenesis, ChIP, and functional transactivation assays; multiple orthogonal methods\",\n      \"pmids\": [\"18521086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CENP-R (ITGB3BP) synergizes with CENP-OPQU to regulate kinetochore-microtubule attachment stability and accurate chromosome segregation in mitosis. Aurora B-mediated phosphorylation of CENP-R weakens its kinetochore localization and disrupts its binding with CENP-U, thereby promoting correction of improper kinetochore-microtubule attachments.\",\n      \"method\": \"Phospho-mimicking mutagenesis, kinetochore localization assays, co-immunoprecipitation (CENP-R/CENP-U interaction), chromosome segregation assays, mitosis imaging\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mutagenesis, co-IP, localization, and functional chromosome segregation readouts combined in one rigorous study\",\n      \"pmids\": [\"36069839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CENP-R (ITGB3BP) is a constitutive kinetochore component throughout the cell cycle in vertebrates and is part of the CENP-O complex (CENP-O/P/Q/R/U). CENP-R-deficient (Cenp-r−/−) mice are viable, indicating it is not essential for normal development, but loss of CENP-R leads to increased early tumor formation and altered apoptosis/proliferation balance in papillomas, suggesting a context-dependent role in cancer progression.\",\n      \"method\": \"Knockout mouse model (Cenp-r−/−), DMBA/TPA chemical carcinogenesis protocol, histological analysis of proliferation and apoptosis markers\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout mouse with defined tumor biology readout, but mechanism is inferred from in vivo phenotype without direct biochemical dissection\",\n      \"pmids\": [\"28795467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CENP-R (ITGB3BP) interacts with EB1 via CENP-R's N-terminal intrinsic disorder region and EB1's end-binding homology domain. EB1 and CENP-R undergo co-condensation (phase separation). An EB1-binding-defective CENP-R mutant perturbs chromosome oscillations, indicating that EB1–CENP-R condensation forms a physical link between the inner kinetochore and dynamic spindle microtubule plus-ends to drive chromosome oscillations in mitosis.\",\n      \"method\": \"NMR interaction mapping, biochemical co-condensation assays, EB1-binding-defective CENP-R mutant expression, live-cell chromosome oscillation imaging\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — NMR structural mapping combined with biochemical condensation assays and functional mutant imaging across orthogonal methods\",\n      \"pmids\": [\"40349345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ITGB3BP protein is subject to ubiquitin-mediated degradation promoted by its interaction with MDM2, as induced by lncRNA NR_045147. ITGB3BP upregulation promotes osteogenic differentiation and migration of periodontal ligament stem cells and enhances mitochondrial respiration.\",\n      \"method\": \"Ubiquitination assay, western blotting for protein stability, osteogenic differentiation assays, migration assays, Seahorse XF mitochondrial respiration analysis, in vivo calvarial defect model\",\n      \"journal\": \"Stem cells translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination assays with functional readouts, single lab, mechanism linked to MDM2 interaction but indirect (via lncRNA context)\",\n      \"pmids\": [\"39674578\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ITGB3BP (NRIF3/TAP20/CENP-R) is a multifunctional protein: as a nuclear receptor coregulator it uses bivalent LXXIL/LXXLL motifs to specifically co-activate TR and RXR (and ERα following Pak1-mediated Ser28 phosphorylation), while its N-terminal RepD1 can repress transcription; it also contains a death domain (DD1) that triggers caspase 2-dependent, mitochondria-involved apoptosis selectively in breast cancer cells; as a structural kinetochore component of the CENP-O complex it is phosphorylated by Aurora B to disrupt CENP-U binding and correct improper kinetochore-microtubule attachments, and co-condenses with EB1 to link the inner kinetochore to dynamic microtubule plus-ends for chromosome oscillations; additionally, it interacts with the β5 integrin cytoplasmic domain downstream of PKCθ to modulate endothelial cell adhesion and migration.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ITGB3BP (NRIF3/TAP20/CENP-R) is a multifunctional protein operating in two largely distinct arenas: nuclear receptor coregulation and mitotic kinetochore function [#0, #6]. As a nuclear coactivator it localizes to the nucleus and uses a bivalent interaction mechanism—a C-terminal LXXIL module (RID1) as the primary receptor-contact and an N-terminal LXXLL motif (RID2) as a modulatory contact—to engage thyroid hormone receptor and RXR dimers in a ligand-dependent manner, with inter-module spacing governing affinity [#0, #1]. Its C-terminus harbors an autonomous transactivation domain while an N-terminal RepD1 (residues 20–50) confers transrepression that is abolished by Ser28Ala, defining this residue as a regulatory phospho-site [#1]. Pak1 phosphorylates Ser28, enhancing nuclear localization, ERα binding, recruitment to ERα target promoters, and estrogen responsiveness, thereby extending coactivator function to ERα [#5]; MTA1 binds the N-terminal LXXLL region and represses NRIF3-driven ERE transcription by blocking its chromatin association [#3]. A separate 30-amino-acid death domain (DD1) drives caspase 2-dependent, mitochondria-involving apoptosis selectively in breast cancer cells [#4]. In mitosis, ITGB3BP is a constitutive kinetochore component of the CENP-O complex (CENP-O/P/Q/R/U) that regulates kinetochore–microtubule attachment stability; Aurora B phosphorylation weakens its kinetochore localization and disrupts its binding to CENP-U to correct erroneous attachments [#6, #7], and co-condensation with EB1 via its N-terminal disordered region links the inner kinetochore to dynamic microtubule plus-ends to drive chromosome oscillations [#8]. Independently, TAP20 acts downstream of PKCθ in endothelial cells, binding the β5 integrin cytoplasmic domain to reduce adhesion and promote migration and tube formation [#2], and its protein levels are controlled by MDM2-promoted ubiquitin-mediated degradation [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established ITGB3BP as a nuclear receptor coactivator with specificity, answering whether it acts broadly or selectively across receptors.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding, mutagenesis and transactivation assays mapping a C-terminal LXXIL (RID1) module\",\n      \"pmids\": [\"10490654\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve stoichiometry of receptor-dimer engagement\", \"No structural model of RID1 binding\", \"Endogenous target genes not defined\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Defined a separate cytoplasmic identity for the same protein, showing it links PKCθ signaling to integrin-dependent endothelial behavior.\",\n      \"evidence\": \"Coprecipitation with β5 integrin cytoplasmic domain plus adhesion, migration, tube-formation and antibody-blocking assays\",\n      \"pmids\": [\"10579726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of integrin-tail binding not mapped\", \"Relationship to the nuclear coactivator role unexplained\", \"Downstream signaling effectors unidentified\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Resolved how the coregulator engages receptors and how its activity is partitioned into activating and repressing domains, defining Ser28 as a regulatory node.\",\n      \"evidence\": \"Deletion/mutagenesis mapping of bivalent RID modules, dimerization domain, AD1, and RepD1 with Gal4 reporter assays\",\n      \"pmids\": [\"11713274\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for Ser28 phosphorylation not yet identified\", \"Physiological context of repression versus activation unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Revealed a coregulatory antagonism, showing MTA1 represses ITGB3BP-driven ER signaling by displacing it from chromatin.\",\n      \"evidence\": \"Y2H, in vitro and in vivo binding, ChIP, and ERE reporter assays\",\n      \"pmids\": [\"15254226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of chromatin displacement not structurally defined\", \"Generality across other receptor target genes untested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified a pro-apoptotic activity distinct from transcription, defining a death domain that selectively kills breast cancer cells.\",\n      \"evidence\": \"Domain mapping, caspase inhibitor studies, and mitochondrial permeability assays in cell lines\",\n      \"pmids\": [\"15082778\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of cell-type selectivity unknown\", \"Upstream trigger of DD1-mediated apoptosis unidentified\", \"Endogenous relevance versus overexpression unclear\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Connected upstream signaling to coregulator output, showing Pak1 phosphorylation of Ser28 activates ITGB3BP toward ERα.\",\n      \"evidence\": \"In vitro kinase assay, phospho-mimic/non-phosphorylatable mutants, ChIP, and transactivation assays\",\n      \"pmids\": [\"18521086\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Interplay between Ser28 activation here and RepD1 repression unresolved\", \"Other phospho-regulated receptors not surveyed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placed ITGB3BP in the constitutive kinetochore CENP-O complex and tested organismal requirement, showing it is dispensable for development but modifies tumor formation.\",\n      \"evidence\": \"Cenp-r−/− knockout mouse with DMBA/TPA carcinogenesis and histological proliferation/apoptosis analysis\",\n      \"pmids\": [\"28795467\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tumor phenotype mechanism inferred from in vivo readout without biochemical dissection\", \"Link between kinetochore role and tumorigenesis not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined how the kinetochore role is regulated, showing Aurora B phosphorylation disengages CENP-R from CENP-U to correct erroneous attachments.\",\n      \"evidence\": \"Phospho-mimic mutagenesis, kinetochore localization, CENP-R/CENP-U co-IP, and chromosome segregation assays\",\n      \"pmids\": [\"36069839\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CENP-R/CENP-U interface not solved\", \"Aurora B phospho-sites not enumerated here\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established a physical mechanism coupling the inner kinetochore to microtubule plus-ends, showing CENP-R co-condenses with EB1 to drive chromosome oscillations.\",\n      \"evidence\": \"NMR interaction mapping, co-condensation assays, and EB1-binding-defective mutant with live-cell oscillation imaging\",\n      \"pmids\": [\"40349345\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulation of the condensation in vivo not defined\", \"Interplay with Aurora B-controlled CENP-U binding unexplored\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified post-translational control of ITGB3BP abundance and a metabolic/differentiation role in stem cells.\",\n      \"evidence\": \"Ubiquitination and stability assays, MDM2 interaction, osteogenic and migration assays, Seahorse respiration, and calvarial defect model\",\n      \"pmids\": [\"39674578\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"MDM2-mediated degradation linked indirectly via lncRNA context\", \"Single lab, mechanism of mitochondrial enhancement undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the protein's nuclear coregulator, apoptotic, kinetochore, and integrin-signaling roles are coordinated within one cell, and whether they share a common regulatory logic, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model linking the distinct functional modules\", \"Isoform/context determinants of which role is active are unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1, 3, 5]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 3, 5]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [6, 8]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [6, 7]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1, 3, 5]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6, 7, 8]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"complexes\": [\"CENP-O complex (CENP-O/P/Q/R/U)\", \"kinetochore\"],\n    \"partners\": [\"THRA\", \"RXRA\", \"ESR1\", \"MTA1\", \"PAK1\", \"ITGB5\", \"CENP-U\", \"EB1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}