{"gene":"RIPOR2","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2012,"finding":"FAM65B (RIPOR2) is a transcriptional target of FOXO1 that binds the small GTPase RhoA via a noncanonical domain and represses RhoA activity by decreasing its GTP loading, thereby negatively regulating chemokine-induced T cell adhesion, morphological polarization, and migration.","method":"Pulldown assays, RhoA activity (GTP-loading) assays, mRNA knockdown and overexpression in T lymphocytes, chemotaxis and adhesion assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding assays, functional GTP-loading assay, loss-of-function and gain-of-function, replicated in subsequent independent studies","pmids":["23241886"],"is_preprint":false},{"year":2014,"finding":"FAM65B (RIPOR2) binds HDAC6 and dysferlin, forming a transient tricomplex (Fam65b–HDAC6–dysferlin) during myogenic cell differentiation; Fam65b expression is necessary for this complex to form, and its knockdown in developing zebrafish causes abnormal muscle with tears at the myosepta.","method":"Protein pulldowns in differentiating human muscle cells and regenerating muscle tissue, HDAC inhibitor treatment, zebrafish knockdown with histological readout","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal pulldowns plus in vivo loss-of-function in two model systems, single lab","pmids":["24687993"],"is_preprint":false},{"year":2014,"finding":"FAM65B (RIPOR2) contains a PX membrane localization domain required for plasma membrane targeting; a splice-site mutation causing deletion of 52 amino acids within this domain results in cytoplasmic inclusion body accumulation instead of membrane localization in stereocilia of cochlear hair cells, and knockdown in zebrafish reduces saccular hair cell and neuromast numbers.","method":"Mutant protein expression and localization (immunofluorescence), zebrafish fam65b morpholino knockdown, cosegregation in consanguineous kindred","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization experiment tied to functional consequence, in vivo loss-of-function in zebrafish, replicated by subsequent hair cell studies","pmids":["24958875"],"is_preprint":false},{"year":2015,"finding":"FAM65B (RIPOR2) accumulates at the leading edge of neutrophils upon chemoattractant stimulation; phosphorylated FAM65B binds 14-3-3 family proteins, increasing its stability, and this accumulation depends on front-signal pathways (PLCβ and PI3Kγ). FAM65B deficiency in neutrophils increases RhoA activity and mislocalizes phosphorylated myosin light chain (pMLC) to the cell front, causing defects in chemotaxis directionality and adhesion to endothelial cells under flow.","method":"Live-cell imaging and subcellular fractionation in neutrophils, phosphorylation assays, Co-IP with 14-3-3 proteins, FAM65B-deficient cells, RhoA activity assays, chemotaxis and adhesion assays under flow","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (localization, phosphorylation, Co-IP, KO phenotype), single lab","pmids":["25588844"],"is_preprint":false},{"year":2016,"finding":"Fam65b (RIPOR2) oligomers form a circumferential ring near the basal taper of stereocilia in murine cochlear hair cells as shown by STORM super-resolution microscopy; RhoC is a binding partner identified by yeast-two-hybrid and co-localizes with Fam65b in stereocilia; RhoC regulates Fam65b oligomerization; oligomerization and RhoC binding are required for Fam65b function. In Fam65b-deficient hair cells, taperin organization is disrupted and mechanotransduction is impaired.","method":"Stochastic optical reconstruction microscopy (STORM), biochemistry (oligomerization assays), yeast two-hybrid, co-localization, Fam65b-knockout mouse mechanotransduction recordings","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — super-resolution structural imaging combined with yeast-two-hybrid, biochemical oligomerization assays, and functional KO electrophysiology in a single rigorous study","pmids":["27269051"],"is_preprint":false},{"year":2016,"finding":"Forced FAM65B (RIPOR2) expression in transformed cells blocks mitosis by disrupting the mitotic spindle, causing G2 arrest and apoptosis; upon proliferation arrest, FAM65B forms a complex with HDAC6 and 14-3-3. In primary T cells, FAM65B is downregulated upon TCR engagement, and maintaining its expression blocks T cell proliferation.","method":"FAM65B overexpression in transformed cell lines, cell cycle analysis, Co-IP (FAM65B–HDAC6–14-3-3 complex), FAM65B knockdown and forced expression in primary T lymphocytes","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP complex identification plus loss/gain-of-function phenotypes, single lab","pmids":["27556504"],"is_preprint":false},{"year":2018,"finding":"Chemokine stimulation phosphorylates Fam65b (RIPOR2) in T lymphocytes; this phosphorylation decreases Fam65b affinity for RhoA and causes Fam65b to shuttle from the plasma membrane to the cytosol. Fam65b-deficient mice show increased spontaneous RhoA activation in resting T cells and defective intranodal T cell migration in vivo. The degree of Fam65b phosphorylation controls actin polymerization downstream of active RhoA and T cell migration in vitro.","method":"Conditional Fam65b-knockout mouse, phosphorylation assays, RhoA binding affinity measurements, subcellular fractionation, intranodal two-photon microscopy, in vitro migration assays","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO mouse with in vivo migration readout, biochemical phosphorylation assays, and membrane/cytosol fractionation; multiple orthogonal methods","pmids":["30254631"],"is_preprint":false},{"year":2018,"finding":"In cardiomyocytes, PINK1 phosphorylates FAM65B (RIPOR2) at serine 46; phosphorylated FAM65B inhibits autophagy and cell death in the heart, placing FAM65B downstream of PINK1 in the ACR circRNA–PINK1–FAM65B axis regulating cardiac autophagy.","method":"In vitro kinase assay (PINK1 phosphorylating FAM65B at S46), cardiac-specific transgenic/overexpression mouse models, I/R injury model, autophagy flux assays","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 1-2 / Weak — kinase assay identifying phosphorylation site, but finding is embedded in a circRNA study; single lab, single paper","pmids":["30349076"],"is_preprint":false},{"year":2018,"finding":"Ripor2 (FAM65B) interacts with Myh9 (non-muscle myosin IIA, encoded by a known deafness gene) in cochlear hair cells; absence of Ripor2 reduces Myh9 protein abundance despite increased Myh9 mRNA, and causes aberrant kinocilium localization associated with reduced phosphorylated Myh9 and reduced acetylated alpha-tubulin in the kinocilium.","method":"Co-immunoprecipitation (Ripor2–Myh9 interaction), western blotting, immunofluorescence in Ripor2-deficient mouse cochlea","journal":"Journal of molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP interaction plus KO phenotype with multiple protein readouts, single lab","pmids":["30280293"],"is_preprint":false},{"year":2006,"finding":"C6ORF32 (RIPOR2) localizes to the cellular cytoskeleton and filopodia in myogenic and non-myogenic cells; overexpression promotes formation of long membrane protrusions (filopodia), and a deletion analysis mapped filopodia-promoting activity to amino acids 55–113. Knockdown in C2C12 myoblasts causes a myogenic differentiation defect with decreased myogenin and myosin heavy chain expression.","method":"Immunofluorescence localization, overexpression with serial deletion mutants, siRNA knockdown in C2C12 cells with myogenic marker western blotting","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — direct localization, domain-mapping by deletions, loss-of-function phenotype; single lab","pmids":["17150207"],"is_preprint":false},{"year":2022,"finding":"Gentamicin binds to RIPOR2 and triggers its rapid translocation from stereocilia to the pericuticular area in murine hair cells; translocated RIPOR2 then interacts with the autophagy component GABARAP to disrupt autophagy. Reducing RIPOR2 or GABARAP expression completely prevents aminoglycoside-induced hair cell death and hearing loss in mice. Abolishing PINK1 or Parkin (mitophagy regulators) also prevents this hair cell death, placing RIPOR2-mediated autophagic dysfunction upstream of mitophagy-driven cell death.","method":"Drug-binding assay (gentamicin–RIPOR2), live-cell imaging of RIPOR2 translocation, Co-IP (RIPOR2–GABARAP interaction), RIPOR2/GABARAP/PINK1/Parkin knockout or knockdown mouse models, ABR hearing threshold measurements","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — drug-binding, Co-IP interaction, and multiple KO/KD mouse models with direct functional hearing readout in a single study","pmids":["36113482"],"is_preprint":false},{"year":2020,"finding":"An in-frame 12-nucleotide deletion in RIPOR2 causes autosomal dominant adult-onset progressive hearing loss; the mutant RIPOR2 protein shows aberrant localization in stereocilia of cochlear hair cells and fails to rescue morphological defects in RIPOR2-deficient hair cells, while wild-type RIPOR2 rescues them, establishing that correct stereociliary localization is functionally necessary.","method":"Ex vivo mutant protein expression and localization in hair cells, rescue assay in RIPOR2-deficient hair cells, exome sequencing co-segregation in 12 families","journal":"Journal of medical genetics","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — direct localization experiment tied to functional rescue assay; single lab but supported by genetic co-segregation","pmids":["32631815"],"is_preprint":false},{"year":2025,"finding":"Aminoglycoside-triggered RIPOR2 translocation and phosphatidylserine externalization in hair cells occur by independent mechanisms; cisplatin and aminoglycosides induce hair cell death via distinct molecular pathways (cisplatin does not replicate aminoglycoside-induced RIPOR2 translocation phenotype).","method":"Time-course live imaging of RIPOR2 translocation and PS externalization in wild-type hair cells with AG treatment, pharmacological and genetic dissection in hair cells","journal":"Frontiers in cellular neuroscience","confidence":"Medium","confidence_rationale":"Tier 2-3 / Weak — direct imaging mechanistic dissection, single lab, single paper","pmids":["40842562"],"is_preprint":false},{"year":2026,"finding":"RIPOR2 is a positive transcriptional target of the RAS/ERK pathway in melanocyte precursors and human melanoma cells; ectopic RIPOR2 expression functionally promotes multinucleation in a chicken embryo in vivo model and in human melanoma-derived cell lines.","method":"Single-nucleus RNA sequencing, RAS/ERK pathway activation in chicken embryo model, RIPOR2 overexpression in human melanoma cell lines, multinucleation quantification","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — in vivo transcriptional target identification combined with functional overexpression in human cells; single lab","pmids":["42100747"],"is_preprint":false},{"year":2025,"finding":"RIPOR2 knockdown in T cells exacerbates exhaustion phenotypes (reduced IFN-γ secretion, increased PD-1); Ginsenoside F3 binds RIPOR2 (molecular docking confirmed), reverses exhaustion phenotypes, and synergizes with anti-PD-1 therapy in a mouse NSCLC model, identifying RIPOR2 as an immunometabolic regulator of T cell exhaustion.","method":"siRNA knockdown of RIPOR2 in in vitro T cell exhaustion model, proteomic profiling, molecular docking, in vivo NSCLC tumor model with anti-PD-1 co-treatment","journal":"Phytomedicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — molecular docking is computational; siRNA phenotype and in vivo data are preliminary, single lab, single paper","pmids":["41418634"],"is_preprint":false}],"current_model":"RIPOR2 (FAM65B) is a multi-functional RhoA inhibitor that binds RhoA via a noncanonical domain to suppress GTP loading; its activity is regulated by phosphorylation (which reduces RhoA affinity and shifts RIPOR2 from plasma membrane to cytosol) and by transcriptional control via FOXO1 and RAS/ERK, placing it at the intersection of cell polarity, migration, and cytoskeletal organization in T lymphocytes, neutrophils, and melanoma cells; in cochlear hair cells, RIPOR2 oligomers form a ring-like structure near the stereociliary base (interacting with RhoC, Myh9, and GABARAP), and its aminoglycoside-triggered translocation from stereocilia dysregulates GABARAP-dependent autophagy/mitophagy to cause hair cell death and hearing loss; in myogenic cells, RIPOR2 forms a transient tricomplex with HDAC6 and dysferlin that is required for myoblast differentiation and fusion."},"narrative":{"mechanistic_narrative":"RIPOR2 (FAM65B) is a RhoA-regulatory cytoskeletal protein that controls cell polarity, migration, and differentiation across multiple cell types [PMID:23241886, PMID:25588844]. It binds the small GTPase RhoA through a noncanonical domain and represses RhoA activity by reducing its GTP loading, thereby negatively regulating chemokine-induced T cell adhesion, polarization, and migration [PMID:23241886]. This activity is regulated by phosphorylation: chemokine-induced phosphorylation lowers RIPOR2 affinity for RhoA and drives its relocation from the plasma membrane to the cytosol, with the degree of phosphorylation tuning RhoA-dependent actin polymerization and T cell migration in vivo [PMID:30254631]. In neutrophils, RIPOR2 accumulates at the leading edge downstream of PLCβ/PI3Kγ signaling, is stabilized by phosphorylation-dependent 14-3-3 binding, and restrains RhoA and phosphorylated myosin light chain at the cell front to maintain chemotactic directionality [PMID:25588844]. Its expression is transcriptionally controlled by FOXO1 [PMID:23241886] and by the RAS/ERK pathway, the latter promoting multinucleation in melanoma cells [PMID:42100747]. In cochlear hair cells, RIPOR2 oligomers form a circumferential ring near the basal taper of stereocilia, binding RhoC (which regulates its oligomerization) and Myh9, and are required for taperin organization and mechanotransduction [PMID:27269051, PMID:30280293]; correct stereociliary localization, dependent on a PX membrane-targeting domain, is functionally essential, and disrupting mutations cause hearing loss [PMID:24958875, PMID:32631815]. Aminoglycosides bind RIPOR2 and trigger its translocation from stereocilia, whereupon it engages the autophagy protein GABARAP to dysregulate autophagy and PINK1/Parkin-dependent mitophagy, driving hair cell death [PMID:36113482]. In myogenic cells RIPOR2 localizes to the cytoskeleton and filopodia and forms a transient HDAC6–dysferlin tricomplex required for myoblast differentiation and fusion [PMID:24687993, PMID:17150207]. RIPOR2 mutation causes autosomal dominant adult-onset progressive hearing loss [PMID:32631815].","teleology":[{"year":2006,"claim":"Established the first cellular role of the protein by linking it to the cytoskeleton and myogenesis, before its RhoA connection was known.","evidence":"Immunofluorescence, overexpression with serial deletion mutants mapping filopodia activity to aa 55–113, and siRNA knockdown in C2C12 myoblasts","pmids":["17150207"],"confidence":"Medium","gaps":["Molecular mechanism linking the protein to actin protrusions not defined","No binding partners identified at this stage"]},{"year":2012,"claim":"Defined the core molecular function as a noncanonical RhoA inhibitor under FOXO1 transcriptional control, answering how it negatively regulates T cell migration.","evidence":"Pulldown and RhoA GTP-loading assays with knockdown/overexpression and chemotaxis/adhesion assays in T lymphocytes","pmids":["23241886"],"confidence":"High","gaps":["Structural basis of the noncanonical RhoA-binding domain not resolved","How RhoA binding is dynamically regulated not yet addressed"]},{"year":2014,"claim":"Connected the protein to muscle differentiation mechanistically by identifying a transient HDAC6–dysferlin tricomplex and an in vivo myogenic requirement.","evidence":"Reciprocal pulldowns in differentiating human muscle, HDAC inhibitor treatment, and zebrafish knockdown with myoseptal histology","pmids":["24687993"],"confidence":"High","gaps":["Function of the tricomplex during fusion not biochemically defined","Whether RhoA regulation participates in myogenesis unknown"]},{"year":2014,"claim":"Identified a PX membrane-targeting domain and tied its disruption to mislocalization and hair cell loss, opening the cochlear/deafness chapter.","evidence":"Mutant protein localization, zebrafish morpholino knockdown, and cosegregation in a consanguineous kindred","pmids":["24958875"],"confidence":"High","gaps":["Lipid specificity of the PX domain not characterized","Stereociliary function of the protein not yet defined"]},{"year":2015,"claim":"Explained how the RhoA inhibitor is spatially controlled at the migrating cell front via phosphorylation-dependent 14-3-3 binding and front-signaling pathways.","evidence":"Live imaging, fractionation, phosphorylation and 14-3-3 Co-IP, and KO chemotaxis/adhesion-under-flow assays in neutrophils","pmids":["25588844"],"confidence":"High","gaps":["Kinase responsible for leading-edge phosphorylation not identified","Quantitative coupling between RhoA suppression and pMLC localization unresolved"]},{"year":2016,"claim":"Resolved the supramolecular organization in stereocilia, identifying RhoC as an oligomerization regulator and linking the protein to mechanotransduction.","evidence":"STORM super-resolution imaging, yeast two-hybrid, oligomerization biochemistry, and KO mouse mechanotransduction recordings","pmids":["27269051"],"confidence":"High","gaps":["Atomic structure of the ring oligomer not determined","Mechanism by which RhoC controls oligomerization not detailed"]},{"year":2016,"claim":"Showed expression level acts as a proliferation/mitotic switch, with a HDAC6–14-3-3 complex forming upon arrest.","evidence":"Overexpression in transformed cells with cell-cycle analysis, Co-IP, and forced expression/knockdown in primary T cells","pmids":["27556504"],"confidence":"Medium","gaps":["Mechanism of spindle disruption not defined","Physiological relevance of mitotic block in non-overexpression contexts unclear"]},{"year":2018,"claim":"Demonstrated phosphorylation as the master switch coupling chemokine signaling to RhoA release and membrane-to-cytosol shuttling in vivo.","evidence":"Conditional KO mouse, phosphorylation and RhoA-affinity assays, fractionation, and intranodal two-photon microscopy","pmids":["30254631"],"confidence":"High","gaps":["Identity of the chemokine-activated kinase not established","Phosphosite mapping not fully resolved in this context"]},{"year":2018,"claim":"Placed the protein in a cardiac autophagy circuit as a PINK1 phosphorylation substrate at S46.","evidence":"In vitro kinase assay, cardiac transgenic mouse models, and I/R injury with autophagy flux assays","pmids":["30349076"],"confidence":"Medium","gaps":["Finding embedded in a circRNA study; direct cardiac function of the protein not independently confirmed","Whether S46 phosphorylation links to RhoA regulation unknown"]},{"year":2018,"claim":"Identified Myh9 as a stereociliary partner and linked the protein to kinocilium positioning and cytoskeletal protein stability.","evidence":"Co-IP, western blotting, and immunofluorescence in Ripor2-deficient mouse cochlea","pmids":["30280293"],"confidence":"Medium","gaps":["Mechanism by which loss reduces Myh9 protein despite higher mRNA unresolved","Direct vs indirect effect on tubulin acetylation not separated"]},{"year":2020,"claim":"Established a human disease link, showing a dominant in-frame deletion causes hearing loss through mislocalization and loss of rescue function.","evidence":"Ex vivo mutant localization, rescue assay in deficient hair cells, and exome co-segregation in 12 families","pmids":["32631815"],"confidence":"Medium","gaps":["Dominant-negative vs haploinsufficiency mechanism not distinguished","Why hearing loss is adult-onset and progressive not explained"]},{"year":2022,"claim":"Defined an ototoxicity mechanism: aminoglycoside binding triggers RIPOR2 translocation that hijacks GABARAP-dependent autophagy and PINK1/Parkin mitophagy to kill hair cells.","evidence":"Gentamicin-binding assay, live imaging, RIPOR2–GABARAP Co-IP, and RIPOR2/GABARAP/PINK1/Parkin KO/KD mice with ABR readouts","pmids":["36113482"],"confidence":"High","gaps":["Structural site of gentamicin binding not mapped","How translocation switches RIPOR2 from cytoskeletal to autophagy function unknown"]},{"year":2025,"claim":"Dissected ototoxic pathways, showing aminoglycoside-induced RIPOR2 translocation is independent of phosphatidylserine externalization and distinct from cisplatin-induced death.","evidence":"Time-course live imaging and pharmacological/genetic dissection in wild-type hair cells","pmids":["40842562"],"confidence":"Medium","gaps":["Molecular trigger separating the two death pathways not identified","Single-lab mechanistic dissection"]},{"year":2026,"claim":"Identified RAS/ERK as a transcriptional driver of RIPOR2 with a functional role in melanoma multinucleation.","evidence":"Single-nucleus RNA-seq, RAS/ERK activation in chicken embryo, and overexpression in human melanoma lines","pmids":["42100747"],"confidence":"Medium","gaps":["Mechanism linking RIPOR2 to multinucleation not defined","Whether RhoA regulation underlies this phenotype untested"]},{"year":null,"claim":"How RIPOR2's distinct functional modes — cytoskeletal RhoA inhibition, stereociliary ring formation, and autophagy regulation — are mechanistically interconverted, and the structural basis of its noncanonical RhoA binding and oligomerization, remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No atomic structure of RIPOR2 or its RhoA/RhoC complexes","Kinases governing context-specific phosphorylation incompletely defined","Unifying principle relating its membrane vs cytosol vs translocated states unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,6]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[9,4]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,10]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,3,6]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6,3]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[9,4]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,6,3]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[10]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[1,9]}],"complexes":["RIPOR2–HDAC6–dysferlin tricomplex"],"partners":["RHOA","RHOC","HDAC6","DYSF","MYH9","GABARAP","YWHAB","PINK1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y4F9","full_name":"Rho family-interacting cell polarization regulator 2","aliases":[],"length_aa":1068,"mass_kda":118.5,"function":"Acts as an inhibitor of the small GTPase RHOA and plays several roles in the regulation of myoblast and hair cell differentiation, lymphocyte T proliferation and neutrophil polarization (PubMed:17150207, PubMed:23241886, PubMed:24687993, PubMed:24958875, PubMed:25588844, PubMed:27556504). Inhibits chemokine-induced T lymphocyte responses, such as cell adhesion, polarization and migration (PubMed:23241886). Involved also in the regulation of neutrophil polarization, chemotaxis and adhesion (By similarity). Required for normal development of inner and outer hair cell stereocilia within the cochlea of the inner ear (By similarity). Plays a role for maintaining the structural organization of the basal domain of stereocilia (By similarity). Involved in mechanosensory hair cell function (By similarity). Required for normal hearing (PubMed:24958875) Acts as an inhibitor of the small GTPase RHOA (PubMed:25588844). Plays a role in fetal mononuclear myoblast differentiation by promoting filopodia and myotube formation (PubMed:17150207). Maintains naive T lymphocytes in a quiescent state (PubMed:27556504)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9Y4F9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RIPOR2","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RIPOR2","total_profiled":1310},"omim":[{"mim_id":"616515","title":"DEAFNESS, AUTOSOMAL RECESSIVE 104; DFNB104","url":"https://www.omim.org/entry/616515"},{"mim_id":"611410","title":"RHO FAMILY-INTERACTING CELL POLARIZATION REGULATOR 2; RIPOR2","url":"https://www.omim.org/entry/611410"},{"mim_id":"607017","title":"DEAFNESS, AUTOSOMAL DOMINANT 21; DFNA21","url":"https://www.omim.org/entry/607017"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"endometrium 1","ntpm":49.3},{"tissue":"lymphoid tissue","ntpm":77.3}],"url":"https://www.proteinatlas.org/search/RIPOR2"},"hgnc":{"alias_symbol":["KIAA0386","DIFF48","MYONAP"],"prev_symbol":["C6orf32","FAM65B"]},"alphafold":{"accession":"Q9Y4F9","domains":[{"cath_id":"-","chopping":"70-197","consensus_level":"high","plddt":91.7797,"start":70,"end":197},{"cath_id":"2.60.40.150","chopping":"202-320","consensus_level":"high","plddt":89.6276,"start":202,"end":320},{"cath_id":"1.25.40","chopping":"919-1051","consensus_level":"medium","plddt":83.9632,"start":919,"end":1051}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y4F9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y4F9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y4F9-F1-predicted_aligned_error_v6.png","plddt_mean":65.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RIPOR2","jax_strain_url":"https://www.jax.org/strain/search?query=RIPOR2"},"sequence":{"accession":"Q9Y4F9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y4F9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y4F9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y4F9"}},"corpus_meta":[{"pmid":"30349076","id":"PMC_30349076","title":"The circular RNA ACR attenuates myocardial ischemia/reperfusion injury by suppressing autophagy via modulation of the Pink1/ FAM65B pathway.","date":"2018","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/30349076","citation_count":221,"is_preprint":false},{"pmid":"24958875","id":"PMC_24958875","title":"FAM65B is a membrane-associated protein of hair cell stereocilia required for hearing.","date":"2014","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/24958875","citation_count":61,"is_preprint":false},{"pmid":"23241886","id":"PMC_23241886","title":"Fam65b is a new transcriptional target of FOXO1 that regulates RhoA signaling for T lymphocyte migration.","date":"2012","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/23241886","citation_count":42,"is_preprint":false},{"pmid":"27269051","id":"PMC_27269051","title":"Murine Fam65b forms ring-like structures at the base of stereocilia critical for mechanosensory hair cell function.","date":"2016","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/27269051","citation_count":36,"is_preprint":false},{"pmid":"17150207","id":"PMC_17150207","title":"C6ORF32 is upregulated during muscle cell differentiation and induces the formation of cellular filopodia.","date":"2006","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/17150207","citation_count":33,"is_preprint":false},{"pmid":"27556504","id":"PMC_27556504","title":"FAM65B controls the proliferation of transformed and primary T cells.","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/27556504","citation_count":29,"is_preprint":false},{"pmid":"24687993","id":"PMC_24687993","title":"Fam65b is important for formation of the HDAC6-dysferlin protein complex during myogenic cell differentiation.","date":"2014","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/24687993","citation_count":28,"is_preprint":false},{"pmid":"30254631","id":"PMC_30254631","title":"Fam65b Phosphorylation Relieves Tonic RhoA Inhibition During T Cell Migration.","date":"2018","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/30254631","citation_count":25,"is_preprint":false},{"pmid":"25588844","id":"PMC_25588844","title":"Front-signal-dependent accumulation of the RHOA inhibitor FAM65B at leading edges polarizes neutrophils.","date":"2015","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/25588844","citation_count":23,"is_preprint":false},{"pmid":"36113482","id":"PMC_36113482","title":"RIPOR2-mediated autophagy dysfunction is critical for aminoglycoside-induced hearing loss.","date":"2022","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/36113482","citation_count":19,"is_preprint":false},{"pmid":"32631815","id":"PMC_32631815","title":"A RIPOR2 in-frame deletion is a frequent and highly penetrant cause of adult-onset hearing loss.","date":"2020","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/32631815","citation_count":17,"is_preprint":false},{"pmid":"30280293","id":"PMC_30280293","title":"Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation.","date":"2018","source":"Journal of molecular medicine (Berlin, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/30280293","citation_count":13,"is_preprint":false},{"pmid":"31218594","id":"PMC_31218594","title":"Faster regeneration associated to high expression of Fam65b and Hdac6 in dysferlin-deficient mouse.","date":"2019","source":"Journal of molecular histology","url":"https://pubmed.ncbi.nlm.nih.gov/31218594","citation_count":6,"is_preprint":false},{"pmid":"30720667","id":"PMC_30720667","title":"Association of FAM65B, AGBL4, and CUX2 genetic polymorphisms with susceptibility to antituberculosis drug-induced hepatotoxicity: validation study in a Chinese Han population.","date":"2019","source":"Pharmacogenetics and genomics","url":"https://pubmed.ncbi.nlm.nih.gov/30720667","citation_count":5,"is_preprint":false},{"pmid":"40087148","id":"PMC_40087148","title":"Maternal Exposure to Environmentally Relevant Concentrations of Tris(2,4-di-tert-butylphenyl) Phosphate-Induced Developmental Toxicity in Zebrafish Offspring via Disrupting foxO1/ripor2 Signaling.","date":"2025","source":"Environmental science & technology","url":"https://pubmed.ncbi.nlm.nih.gov/40087148","citation_count":5,"is_preprint":false},{"pmid":"41418634","id":"PMC_41418634","title":"Ginsenoside F3 alleviates T cell exhaustion via RIPOR2-mediated immunometabolic reprogramming to potentiate anti-PD-1 therapy.","date":"2025","source":"Phytomedicine : international journal of phytotherapy and phytopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/41418634","citation_count":0,"is_preprint":false},{"pmid":"40842562","id":"PMC_40842562","title":"Aminoglycoside induces RIPOR2 translocation and phosphatidylserine externalization via distinct mechanisms.","date":"2025","source":"Frontiers in cellular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/40842562","citation_count":0,"is_preprint":false},{"pmid":"37864412","id":"PMC_37864412","title":"RIPOR2: A new gene of non-syndromic cochleovestibular dysfunction, discrepancy between human pathology and animal models.","date":"2023","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/37864412","citation_count":0,"is_preprint":false},{"pmid":"42100747","id":"PMC_42100747","title":"RIPOR2 promotes multinucleation of melanoma cells downstream of the RAS/ERK oncogenic pathway.","date":"2026","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/42100747","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.06.25.661639","title":"Quantitative proteomics and phosphoproteomics reveal glucocorticoid stimulation of TLR and Rho GTPase signaling in neutrophil-like cells","date":"2025-06-26","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.25.661639","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10926,"output_tokens":4193,"usd":0.047836,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12161,"output_tokens":4547,"usd":0.08724,"stage2_stop_reason":"end_turn"},"total_usd":0.135076,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"FAM65B (RIPOR2) is a transcriptional target of FOXO1 that binds the small GTPase RhoA via a noncanonical domain and represses RhoA activity by decreasing its GTP loading, thereby negatively regulating chemokine-induced T cell adhesion, morphological polarization, and migration.\",\n      \"method\": \"Pulldown assays, RhoA activity (GTP-loading) assays, mRNA knockdown and overexpression in T lymphocytes, chemotaxis and adhesion assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding assays, functional GTP-loading assay, loss-of-function and gain-of-function, replicated in subsequent independent studies\",\n      \"pmids\": [\"23241886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"FAM65B (RIPOR2) binds HDAC6 and dysferlin, forming a transient tricomplex (Fam65b–HDAC6–dysferlin) during myogenic cell differentiation; Fam65b expression is necessary for this complex to form, and its knockdown in developing zebrafish causes abnormal muscle with tears at the myosepta.\",\n      \"method\": \"Protein pulldowns in differentiating human muscle cells and regenerating muscle tissue, HDAC inhibitor treatment, zebrafish knockdown with histological readout\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal pulldowns plus in vivo loss-of-function in two model systems, single lab\",\n      \"pmids\": [\"24687993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"FAM65B (RIPOR2) contains a PX membrane localization domain required for plasma membrane targeting; a splice-site mutation causing deletion of 52 amino acids within this domain results in cytoplasmic inclusion body accumulation instead of membrane localization in stereocilia of cochlear hair cells, and knockdown in zebrafish reduces saccular hair cell and neuromast numbers.\",\n      \"method\": \"Mutant protein expression and localization (immunofluorescence), zebrafish fam65b morpholino knockdown, cosegregation in consanguineous kindred\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization experiment tied to functional consequence, in vivo loss-of-function in zebrafish, replicated by subsequent hair cell studies\",\n      \"pmids\": [\"24958875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"FAM65B (RIPOR2) accumulates at the leading edge of neutrophils upon chemoattractant stimulation; phosphorylated FAM65B binds 14-3-3 family proteins, increasing its stability, and this accumulation depends on front-signal pathways (PLCβ and PI3Kγ). FAM65B deficiency in neutrophils increases RhoA activity and mislocalizes phosphorylated myosin light chain (pMLC) to the cell front, causing defects in chemotaxis directionality and adhesion to endothelial cells under flow.\",\n      \"method\": \"Live-cell imaging and subcellular fractionation in neutrophils, phosphorylation assays, Co-IP with 14-3-3 proteins, FAM65B-deficient cells, RhoA activity assays, chemotaxis and adhesion assays under flow\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (localization, phosphorylation, Co-IP, KO phenotype), single lab\",\n      \"pmids\": [\"25588844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Fam65b (RIPOR2) oligomers form a circumferential ring near the basal taper of stereocilia in murine cochlear hair cells as shown by STORM super-resolution microscopy; RhoC is a binding partner identified by yeast-two-hybrid and co-localizes with Fam65b in stereocilia; RhoC regulates Fam65b oligomerization; oligomerization and RhoC binding are required for Fam65b function. In Fam65b-deficient hair cells, taperin organization is disrupted and mechanotransduction is impaired.\",\n      \"method\": \"Stochastic optical reconstruction microscopy (STORM), biochemistry (oligomerization assays), yeast two-hybrid, co-localization, Fam65b-knockout mouse mechanotransduction recordings\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — super-resolution structural imaging combined with yeast-two-hybrid, biochemical oligomerization assays, and functional KO electrophysiology in a single rigorous study\",\n      \"pmids\": [\"27269051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Forced FAM65B (RIPOR2) expression in transformed cells blocks mitosis by disrupting the mitotic spindle, causing G2 arrest and apoptosis; upon proliferation arrest, FAM65B forms a complex with HDAC6 and 14-3-3. In primary T cells, FAM65B is downregulated upon TCR engagement, and maintaining its expression blocks T cell proliferation.\",\n      \"method\": \"FAM65B overexpression in transformed cell lines, cell cycle analysis, Co-IP (FAM65B–HDAC6–14-3-3 complex), FAM65B knockdown and forced expression in primary T lymphocytes\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP complex identification plus loss/gain-of-function phenotypes, single lab\",\n      \"pmids\": [\"27556504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Chemokine stimulation phosphorylates Fam65b (RIPOR2) in T lymphocytes; this phosphorylation decreases Fam65b affinity for RhoA and causes Fam65b to shuttle from the plasma membrane to the cytosol. Fam65b-deficient mice show increased spontaneous RhoA activation in resting T cells and defective intranodal T cell migration in vivo. The degree of Fam65b phosphorylation controls actin polymerization downstream of active RhoA and T cell migration in vitro.\",\n      \"method\": \"Conditional Fam65b-knockout mouse, phosphorylation assays, RhoA binding affinity measurements, subcellular fractionation, intranodal two-photon microscopy, in vitro migration assays\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO mouse with in vivo migration readout, biochemical phosphorylation assays, and membrane/cytosol fractionation; multiple orthogonal methods\",\n      \"pmids\": [\"30254631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In cardiomyocytes, PINK1 phosphorylates FAM65B (RIPOR2) at serine 46; phosphorylated FAM65B inhibits autophagy and cell death in the heart, placing FAM65B downstream of PINK1 in the ACR circRNA–PINK1–FAM65B axis regulating cardiac autophagy.\",\n      \"method\": \"In vitro kinase assay (PINK1 phosphorylating FAM65B at S46), cardiac-specific transgenic/overexpression mouse models, I/R injury model, autophagy flux assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Weak — kinase assay identifying phosphorylation site, but finding is embedded in a circRNA study; single lab, single paper\",\n      \"pmids\": [\"30349076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Ripor2 (FAM65B) interacts with Myh9 (non-muscle myosin IIA, encoded by a known deafness gene) in cochlear hair cells; absence of Ripor2 reduces Myh9 protein abundance despite increased Myh9 mRNA, and causes aberrant kinocilium localization associated with reduced phosphorylated Myh9 and reduced acetylated alpha-tubulin in the kinocilium.\",\n      \"method\": \"Co-immunoprecipitation (Ripor2–Myh9 interaction), western blotting, immunofluorescence in Ripor2-deficient mouse cochlea\",\n      \"journal\": \"Journal of molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP interaction plus KO phenotype with multiple protein readouts, single lab\",\n      \"pmids\": [\"30280293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"C6ORF32 (RIPOR2) localizes to the cellular cytoskeleton and filopodia in myogenic and non-myogenic cells; overexpression promotes formation of long membrane protrusions (filopodia), and a deletion analysis mapped filopodia-promoting activity to amino acids 55–113. Knockdown in C2C12 myoblasts causes a myogenic differentiation defect with decreased myogenin and myosin heavy chain expression.\",\n      \"method\": \"Immunofluorescence localization, overexpression with serial deletion mutants, siRNA knockdown in C2C12 cells with myogenic marker western blotting\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — direct localization, domain-mapping by deletions, loss-of-function phenotype; single lab\",\n      \"pmids\": [\"17150207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Gentamicin binds to RIPOR2 and triggers its rapid translocation from stereocilia to the pericuticular area in murine hair cells; translocated RIPOR2 then interacts with the autophagy component GABARAP to disrupt autophagy. Reducing RIPOR2 or GABARAP expression completely prevents aminoglycoside-induced hair cell death and hearing loss in mice. Abolishing PINK1 or Parkin (mitophagy regulators) also prevents this hair cell death, placing RIPOR2-mediated autophagic dysfunction upstream of mitophagy-driven cell death.\",\n      \"method\": \"Drug-binding assay (gentamicin–RIPOR2), live-cell imaging of RIPOR2 translocation, Co-IP (RIPOR2–GABARAP interaction), RIPOR2/GABARAP/PINK1/Parkin knockout or knockdown mouse models, ABR hearing threshold measurements\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — drug-binding, Co-IP interaction, and multiple KO/KD mouse models with direct functional hearing readout in a single study\",\n      \"pmids\": [\"36113482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"An in-frame 12-nucleotide deletion in RIPOR2 causes autosomal dominant adult-onset progressive hearing loss; the mutant RIPOR2 protein shows aberrant localization in stereocilia of cochlear hair cells and fails to rescue morphological defects in RIPOR2-deficient hair cells, while wild-type RIPOR2 rescues them, establishing that correct stereociliary localization is functionally necessary.\",\n      \"method\": \"Ex vivo mutant protein expression and localization in hair cells, rescue assay in RIPOR2-deficient hair cells, exome sequencing co-segregation in 12 families\",\n      \"journal\": \"Journal of medical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — direct localization experiment tied to functional rescue assay; single lab but supported by genetic co-segregation\",\n      \"pmids\": [\"32631815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Aminoglycoside-triggered RIPOR2 translocation and phosphatidylserine externalization in hair cells occur by independent mechanisms; cisplatin and aminoglycosides induce hair cell death via distinct molecular pathways (cisplatin does not replicate aminoglycoside-induced RIPOR2 translocation phenotype).\",\n      \"method\": \"Time-course live imaging of RIPOR2 translocation and PS externalization in wild-type hair cells with AG treatment, pharmacological and genetic dissection in hair cells\",\n      \"journal\": \"Frontiers in cellular neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Weak — direct imaging mechanistic dissection, single lab, single paper\",\n      \"pmids\": [\"40842562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RIPOR2 is a positive transcriptional target of the RAS/ERK pathway in melanocyte precursors and human melanoma cells; ectopic RIPOR2 expression functionally promotes multinucleation in a chicken embryo in vivo model and in human melanoma-derived cell lines.\",\n      \"method\": \"Single-nucleus RNA sequencing, RAS/ERK pathway activation in chicken embryo model, RIPOR2 overexpression in human melanoma cell lines, multinucleation quantification\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — in vivo transcriptional target identification combined with functional overexpression in human cells; single lab\",\n      \"pmids\": [\"42100747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RIPOR2 knockdown in T cells exacerbates exhaustion phenotypes (reduced IFN-γ secretion, increased PD-1); Ginsenoside F3 binds RIPOR2 (molecular docking confirmed), reverses exhaustion phenotypes, and synergizes with anti-PD-1 therapy in a mouse NSCLC model, identifying RIPOR2 as an immunometabolic regulator of T cell exhaustion.\",\n      \"method\": \"siRNA knockdown of RIPOR2 in in vitro T cell exhaustion model, proteomic profiling, molecular docking, in vivo NSCLC tumor model with anti-PD-1 co-treatment\",\n      \"journal\": \"Phytomedicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — molecular docking is computational; siRNA phenotype and in vivo data are preliminary, single lab, single paper\",\n      \"pmids\": [\"41418634\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RIPOR2 (FAM65B) is a multi-functional RhoA inhibitor that binds RhoA via a noncanonical domain to suppress GTP loading; its activity is regulated by phosphorylation (which reduces RhoA affinity and shifts RIPOR2 from plasma membrane to cytosol) and by transcriptional control via FOXO1 and RAS/ERK, placing it at the intersection of cell polarity, migration, and cytoskeletal organization in T lymphocytes, neutrophils, and melanoma cells; in cochlear hair cells, RIPOR2 oligomers form a ring-like structure near the stereociliary base (interacting with RhoC, Myh9, and GABARAP), and its aminoglycoside-triggered translocation from stereocilia dysregulates GABARAP-dependent autophagy/mitophagy to cause hair cell death and hearing loss; in myogenic cells, RIPOR2 forms a transient tricomplex with HDAC6 and dysferlin that is required for myoblast differentiation and fusion.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RIPOR2 (FAM65B) is a RhoA-regulatory cytoskeletal protein that controls cell polarity, migration, and differentiation across multiple cell types [#0, #3]. It binds the small GTPase RhoA through a noncanonical domain and represses RhoA activity by reducing its GTP loading, thereby negatively regulating chemokine-induced T cell adhesion, polarization, and migration [#0]. This activity is regulated by phosphorylation: chemokine-induced phosphorylation lowers RIPOR2 affinity for RhoA and drives its relocation from the plasma membrane to the cytosol, with the degree of phosphorylation tuning RhoA-dependent actin polymerization and T cell migration in vivo [#6]. In neutrophils, RIPOR2 accumulates at the leading edge downstream of PLC\\u03b2/PI3K\\u03b3 signaling, is stabilized by phosphorylation-dependent 14-3-3 binding, and restrains RhoA and phosphorylated myosin light chain at the cell front to maintain chemotactic directionality [#3]. Its expression is transcriptionally controlled by FOXO1 [#0] and by the RAS/ERK pathway, the latter promoting multinucleation in melanoma cells [#13]. In cochlear hair cells, RIPOR2 oligomers form a circumferential ring near the basal taper of stereocilia, binding RhoC (which regulates its oligomerization) and Myh9, and are required for taperin organization and mechanotransduction [#4, #8]; correct stereociliary localization, dependent on a PX membrane-targeting domain, is functionally essential, and disrupting mutations cause hearing loss [#2, #11]. Aminoglycosides bind RIPOR2 and trigger its translocation from stereocilia, whereupon it engages the autophagy protein GABARAP to dysregulate autophagy and PINK1/Parkin-dependent mitophagy, driving hair cell death [#10]. In myogenic cells RIPOR2 localizes to the cytoskeleton and filopodia and forms a transient HDAC6\\u2013dysferlin tricomplex required for myoblast differentiation and fusion [#1, #9]. RIPOR2 mutation causes autosomal dominant adult-onset progressive hearing loss [#11].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established the first cellular role of the protein by linking it to the cytoskeleton and myogenesis, before its RhoA connection was known.\",\n      \"evidence\": \"Immunofluorescence, overexpression with serial deletion mutants mapping filopodia activity to aa 55\\u2013113, and siRNA knockdown in C2C12 myoblasts\",\n      \"pmids\": [\"17150207\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism linking the protein to actin protrusions not defined\", \"No binding partners identified at this stage\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the core molecular function as a noncanonical RhoA inhibitor under FOXO1 transcriptional control, answering how it negatively regulates T cell migration.\",\n      \"evidence\": \"Pulldown and RhoA GTP-loading assays with knockdown/overexpression and chemotaxis/adhesion assays in T lymphocytes\",\n      \"pmids\": [\"23241886\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the noncanonical RhoA-binding domain not resolved\", \"How RhoA binding is dynamically regulated not yet addressed\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected the protein to muscle differentiation mechanistically by identifying a transient HDAC6\\u2013dysferlin tricomplex and an in vivo myogenic requirement.\",\n      \"evidence\": \"Reciprocal pulldowns in differentiating human muscle, HDAC inhibitor treatment, and zebrafish knockdown with myoseptal histology\",\n      \"pmids\": [\"24687993\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Function of the tricomplex during fusion not biochemically defined\", \"Whether RhoA regulation participates in myogenesis unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified a PX membrane-targeting domain and tied its disruption to mislocalization and hair cell loss, opening the cochlear/deafness chapter.\",\n      \"evidence\": \"Mutant protein localization, zebrafish morpholino knockdown, and cosegregation in a consanguineous kindred\",\n      \"pmids\": [\"24958875\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Lipid specificity of the PX domain not characterized\", \"Stereociliary function of the protein not yet defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Explained how the RhoA inhibitor is spatially controlled at the migrating cell front via phosphorylation-dependent 14-3-3 binding and front-signaling pathways.\",\n      \"evidence\": \"Live imaging, fractionation, phosphorylation and 14-3-3 Co-IP, and KO chemotaxis/adhesion-under-flow assays in neutrophils\",\n      \"pmids\": [\"25588844\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for leading-edge phosphorylation not identified\", \"Quantitative coupling between RhoA suppression and pMLC localization unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved the supramolecular organization in stereocilia, identifying RhoC as an oligomerization regulator and linking the protein to mechanotransduction.\",\n      \"evidence\": \"STORM super-resolution imaging, yeast two-hybrid, oligomerization biochemistry, and KO mouse mechanotransduction recordings\",\n      \"pmids\": [\"27269051\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of the ring oligomer not determined\", \"Mechanism by which RhoC controls oligomerization not detailed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed expression level acts as a proliferation/mitotic switch, with a HDAC6\\u201314-3-3 complex forming upon arrest.\",\n      \"evidence\": \"Overexpression in transformed cells with cell-cycle analysis, Co-IP, and forced expression/knockdown in primary T cells\",\n      \"pmids\": [\"27556504\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of spindle disruption not defined\", \"Physiological relevance of mitotic block in non-overexpression contexts unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated phosphorylation as the master switch coupling chemokine signaling to RhoA release and membrane-to-cytosol shuttling in vivo.\",\n      \"evidence\": \"Conditional KO mouse, phosphorylation and RhoA-affinity assays, fractionation, and intranodal two-photon microscopy\",\n      \"pmids\": [\"30254631\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the chemokine-activated kinase not established\", \"Phosphosite mapping not fully resolved in this context\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Placed the protein in a cardiac autophagy circuit as a PINK1 phosphorylation substrate at S46.\",\n      \"evidence\": \"In vitro kinase assay, cardiac transgenic mouse models, and I/R injury with autophagy flux assays\",\n      \"pmids\": [\"30349076\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Finding embedded in a circRNA study; direct cardiac function of the protein not independently confirmed\", \"Whether S46 phosphorylation links to RhoA regulation unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified Myh9 as a stereociliary partner and linked the protein to kinocilium positioning and cytoskeletal protein stability.\",\n      \"evidence\": \"Co-IP, western blotting, and immunofluorescence in Ripor2-deficient mouse cochlea\",\n      \"pmids\": [\"30280293\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which loss reduces Myh9 protein despite higher mRNA unresolved\", \"Direct vs indirect effect on tubulin acetylation not separated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established a human disease link, showing a dominant in-frame deletion causes hearing loss through mislocalization and loss of rescue function.\",\n      \"evidence\": \"Ex vivo mutant localization, rescue assay in deficient hair cells, and exome co-segregation in 12 families\",\n      \"pmids\": [\"32631815\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Dominant-negative vs haploinsufficiency mechanism not distinguished\", \"Why hearing loss is adult-onset and progressive not explained\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined an ototoxicity mechanism: aminoglycoside binding triggers RIPOR2 translocation that hijacks GABARAP-dependent autophagy and PINK1/Parkin mitophagy to kill hair cells.\",\n      \"evidence\": \"Gentamicin-binding assay, live imaging, RIPOR2\\u2013GABARAP Co-IP, and RIPOR2/GABARAP/PINK1/Parkin KO/KD mice with ABR readouts\",\n      \"pmids\": [\"36113482\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural site of gentamicin binding not mapped\", \"How translocation switches RIPOR2 from cytoskeletal to autophagy function unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Dissected ototoxic pathways, showing aminoglycoside-induced RIPOR2 translocation is independent of phosphatidylserine externalization and distinct from cisplatin-induced death.\",\n      \"evidence\": \"Time-course live imaging and pharmacological/genetic dissection in wild-type hair cells\",\n      \"pmids\": [\"40842562\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular trigger separating the two death pathways not identified\", \"Single-lab mechanistic dissection\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified RAS/ERK as a transcriptional driver of RIPOR2 with a functional role in melanoma multinucleation.\",\n      \"evidence\": \"Single-nucleus RNA-seq, RAS/ERK activation in chicken embryo, and overexpression in human melanoma lines\",\n      \"pmids\": [\"42100747\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking RIPOR2 to multinucleation not defined\", \"Whether RhoA regulation underlies this phenotype untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RIPOR2's distinct functional modes \\u2014 cytoskeletal RhoA inhibition, stereociliary ring formation, and autophagy regulation \\u2014 are mechanistically interconverted, and the structural basis of its noncanonical RhoA binding and oligomerization, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No atomic structure of RIPOR2 or its RhoA/RhoC complexes\", \"Kinases governing context-specific phosphorylation incompletely defined\", \"Unifying principle relating its membrane vs cytosol vs translocated states unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [9, 4]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 3, 6]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6, 3]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [9, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 6, 3]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 9]}\n    ],\n    \"complexes\": [\n      \"RIPOR2\\u2013HDAC6\\u2013dysferlin tricomplex\"\n    ],\n    \"partners\": [\n      \"RHOA\",\n      \"RHOC\",\n      \"HDAC6\",\n      \"DYSF\",\n      \"MYH9\",\n      \"GABARAP\",\n      \"YWHAB\",\n      \"PINK1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":9,"faith_pct":88.88888888888889}}