{"gene":"ARHGEF5","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2011,"finding":"ARHGEF5 (a Dbl-family Rho-GEF) was identified as a Src SH3-domain binding protein required for Src-induced podosome formation. RNAi depletion of ARHGEF5 robustly inhibited podosome formation. ARHGEF5 activates RhoA and Cdc42, is tyrosine-phosphorylated by Src, positively regulates Src kinase activity, and its PH domain is required for podosome formation. ARHGEF5 also forms a ternary complex with Src and PI3K upon Src/ARHGEF5 upregulation.","method":"Co-immunoprecipitation (Src SH3 pulldown), RNAi knockdown with podosome formation assay, overexpression with RhoA/Cdc42 activation assays, tyrosine phosphorylation assay, domain deletion/mutation analysis","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (pulldown, RNAi phenotype, kinase assay, domain mutants) in a single study with clear mechanistic readouts","pmids":["21525037"],"is_preprint":false},{"year":2009,"finding":"ARHGEF5 strongly activates RhoA and RhoB, and weakly RhoC and RhoG, but not Rac1, RhoQ, RhoD, or RhoV, as determined in HEK293 transfection assays. Gβγ subunits interact with ARHGEF5 and stimulate ARHGEF5-mediated RhoA activation in vitro. In vivo, ARHGEF5 deficiency (knockout mice) selectively abrogated MIP1α-induced chemotaxis of immature dendritic cells and impaired DC migration from skin to lymph node, while chemotaxis of macrophages, T/B lymphocytes, and mature DCs was unaffected.","method":"In vitro RhoGTPase activation assay (transfection-based), Gβγ co-immunoprecipitation, in vitro GEF assay, Arhgef5-null mouse with DC chemotaxis and skin-to-lymph-node migration assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — in vitro GEF assay defining substrate specificity, Gβγ interaction, and clean KO mouse with specific cellular phenotype (immature DC chemotaxis)","pmids":["19713215"],"is_preprint":false},{"year":2016,"finding":"ARHGEF5 is upregulated during TGF-β-induced epithelial-mesenchymal transition (EMT) in MCF10A cells and promotes cell migration via the Rho-ROCK pathway. ARHGEF5 is required for in vitro invasion and in vivo metastasis of HCT116 colorectal cancer cells. In mesenchymal-like cells, ARHGEF5 activates Akt via the PI3K pathway to promote tumor growth, a dependence not seen in epithelial-like cells. TNF-α or Slug-induced EMT in HCT116 cells rendered tumor growth dependent on ARHGEF5.","method":"siRNA knockdown, overexpression, in vitro invasion assays, in vivo xenograft/metastasis assay, Akt/PI3K pathway analysis, EMT induction with TGF-β/TNF-α/Slug","journal":"Oncogenesis","confidence":"Medium","confidence_rationale":"Tier 2 — KO/KD with specific cellular and in vivo phenotypes, pathway placement via Rho-ROCK and PI3K-Akt, single lab","pmids":["27617642"],"is_preprint":false},{"year":2015,"finding":"ARHGEF5 (short isoform also called TIM) contains an auto-inhibitory mechanism in which a putative helix N-terminal to the DH domain is stabilized by intramolecular interaction between the C-terminal SH3 domain and a poly-proline sequence between the helix and the DH domain. Designed peptide aptamers that competitively bind the SH3 domain relieve auto-inhibition and activate TIM-catalyzed RhoA guanine nucleotide exchange in vitro. Mutation of Pro49, Pro52, or Lys54 in the designed peptide abolished SH3 binding and GEF activation.","method":"Molecular dynamics simulation, in vitro GEF (guanine nucleotide exchange) assay, peptide binding assay, site-directed mutagenesis of peptide aptamers","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 1-2 — in vitro GEF assay with mutagenesis validating auto-inhibitory mechanism; single lab, computational model supported by biochemical validation","pmids":["25645980"],"is_preprint":false}],"current_model":"ARHGEF5 is a Dbl-family Rho guanine nucleotide exchange factor that preferentially activates RhoA/RhoB, is held in an auto-inhibited state via an intramolecular SH3–polyproline interaction, is relieved of inhibition and activated by Src-mediated tyrosine phosphorylation and Gβγ binding, and drives Src-induced podosome formation, immature dendritic cell chemotaxis, and EMT-associated tumor invasion/metastasis through the Rho-ROCK and PI3K-Akt pathways."},"narrative":{"teleology":[{"year":2009,"claim":"Defining substrate specificity and in vivo function: ARHGEF5 was shown to be a RhoA/RhoB-selective GEF stimulated by Gβγ subunits, and Arhgef5-null mice revealed a non-redundant requirement for immature dendritic cell chemotaxis and skin-to-lymph-node migration, establishing a specific physiological role for this exchange factor.","evidence":"In vitro GEF assays with panel of Rho GTPases, Gβγ co-immunoprecipitation, and Arhgef5-knockout mouse DC chemotaxis and migration assays","pmids":["19713215"],"confidence":"High","gaps":["Mechanism by which Gβγ relieves auto-inhibition is not structurally resolved","Whether ARHGEF5 functions in mature DC or other leukocyte migration under different stimuli is untested","No crystal structure of the ARHGEF5-RhoA catalytic complex"]},{"year":2011,"claim":"Linking ARHGEF5 to Src signaling and podosomes: ARHGEF5 was identified as a Src SH3-domain partner that is tyrosine-phosphorylated by Src, positively regulates Src activity, and is required for Src-induced podosome formation through its PH domain and a ternary complex with Src and PI3K.","evidence":"Src SH3 pulldown, RNAi knockdown with podosome quantification, tyrosine phosphorylation assays, domain deletion/mutation analysis","pmids":["21525037"],"confidence":"High","gaps":["Identity of the specific tyrosine residue(s) phosphorylated by Src is not mapped","How positive feedback between ARHGEF5 and Src kinase activity is regulated to avoid runaway signaling is unclear","Relative contributions of RhoA versus Cdc42 activation downstream of ARHGEF5 in podosome assembly are not dissected"]},{"year":2015,"claim":"Elucidating the auto-inhibitory mechanism: an intramolecular SH3–polyproline interaction was shown to stabilize an inhibitory helix N-terminal to the DH domain, and competitive disruption of this interaction by designed peptide aptamers activated RhoA exchange in vitro, providing a molecular basis for regulation of ARHGEF5 catalytic activity.","evidence":"Molecular dynamics simulation, in vitro GEF assay with peptide aptamers, site-directed mutagenesis of peptide residues","pmids":["25645980"],"confidence":"Medium","gaps":["No experimental structure (crystal or cryo-EM) of the auto-inhibited full-length protein","Whether Src phosphorylation or Gβγ binding directly disrupts the SH3–polyproline interaction in cells is not demonstrated","In vivo validation of the peptide-aptamer activation approach is lacking"]},{"year":2016,"claim":"Establishing a pro-metastatic role in EMT: ARHGEF5 was found to be upregulated during TGF-β-induced EMT and to be required for invasion and in vivo metastasis of colorectal cancer cells, operating through Rho-ROCK and PI3K-Akt signaling axes with mesenchymal-state selectivity.","evidence":"siRNA knockdown and overexpression in MCF10A and HCT116 cells, in vitro invasion assays, in vivo xenograft/metastasis models, EMT induction with TGF-β/TNF-α/Slug","pmids":["27617642"],"confidence":"Medium","gaps":["Transcriptional mechanism of ARHGEF5 upregulation during EMT is not defined","Whether ARHGEF5 dependence generalizes beyond HCT116 and MCF10A models is untested","Direct versus indirect activation of PI3K-Akt by ARHGEF5 in mesenchymal cells is unresolved"]},{"year":null,"claim":"A full structural understanding of how Src phosphorylation, Gβγ binding, and the intramolecular SH3–polyproline switch converge to control ARHGEF5 activation remains unresolved, and additional in vivo roles beyond DC chemotaxis and cancer metastasis have not been explored.","evidence":"","pmids":[],"confidence":"High","gaps":["No high-resolution structure of full-length ARHGEF5 in auto-inhibited or active states","Phosphosite-specific resolution of Src-mediated activation is missing","Potential roles in other Gβγ-coupled immune or developmental processes are unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,3]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1]}],"complexes":[],"partners":["SRC","PIK3CA","GNB1","RHOA","RHOB"],"other_free_text":[]},"mechanistic_narrative":"ARHGEF5 is a Dbl-family Rho guanine nucleotide exchange factor that preferentially catalyzes nucleotide exchange on RhoA and RhoB, with weak activity toward RhoC and RhoG, and is held in an auto-inhibited state by an intramolecular interaction between its C-terminal SH3 domain and an internal polyproline motif that stabilizes an inhibitory helix N-terminal to the DH domain [PMID:25645980, PMID:19713215]. Relief of auto-inhibition occurs through Src-mediated tyrosine phosphorylation and Gβγ binding, enabling ARHGEF5 to drive Src-induced podosome formation via a ternary complex with Src and PI3K, and to support MIP1α-directed chemotaxis of immature dendritic cells as demonstrated by selective loss of this migration in Arhgef5-knockout mice [PMID:21525037, PMID:19713215]. ARHGEF5 is upregulated during TGF-β-induced epithelial–mesenchymal transition and promotes tumor cell invasion and metastasis through the Rho-ROCK and PI3K-Akt pathways, with mesenchymal-like cancer cells becoming selectively dependent on ARHGEF5 for Akt activation and tumor growth [PMID:27617642]."},"prefetch_data":{"uniprot":{"accession":"Q12774","full_name":"Rho guanine nucleotide exchange factor 5","aliases":["Ephexin-3","Guanine nucleotide regulatory protein TIM","Oncogene TIM","Transforming immortalized mammary oncogene","p60 TIM"],"length_aa":1597,"mass_kda":176.8,"function":"Guanine nucleotide exchange factor which activates Rho GTPases (PubMed:15601624). Strongly activates RHOA (PubMed:15601624). Also strongly activates RHOB, weakly activates RHOC and RHOG and shows no effect on RHOD, RHOV, RHOQ or RAC1 (By similarity). Involved in regulation of cell shape and actin cytoskeletal organization (PubMed:15601624). Plays a role in actin organization by generating a loss of actin stress fibers and the formation of membrane ruffles and filopodia (PubMed:14662653). Required for SRC-induced podosome formation (By similarity). Involved in positive regulation of immature dendritic cell migration (By similarity)","subcellular_location":"Cytoplasm; Nucleus; Cell projection, podosome","url":"https://www.uniprot.org/uniprotkb/Q12774/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ARHGEF5","classification":"Not Classified","n_dependent_lines":331,"n_total_lines":1208,"dependency_fraction":0.2740066225165563},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ARHGEF5","total_profiled":1310},"omim":[{"mim_id":"612496","title":"RHO GUANINE NUCLEOTIDE EXCHANGE FACTOR 19; ARHGEF19","url":"https://www.omim.org/entry/612496"},{"mim_id":"608504","title":"RHO GUANINE NUCLEOTIDE EXCHANGE FACTOR 15; ARHGEF15","url":"https://www.omim.org/entry/608504"},{"mim_id":"600888","title":"RHO GUANINE NUCLEOTIDE EXCHANGE FACTOR 5; ARHGEF5","url":"https://www.omim.org/entry/600888"},{"mim_id":"114480","title":"BREAST CANCER","url":"https://www.omim.org/entry/114480"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"skin 1","ntpm":45.2}],"url":"https://www.proteinatlas.org/search/ARHGEF5"},"hgnc":{"alias_symbol":["TIM","TIM1","GEF5","P60"],"prev_symbol":[]},"alphafold":{"accession":"Q12774","domains":[{"cath_id":"1.20.900.10","chopping":"1098-1105_1150-1358","consensus_level":"high","plddt":91.9613,"start":1098,"end":1358},{"cath_id":"2.30.29.30","chopping":"1379-1405_1412-1501","consensus_level":"medium","plddt":86.7192,"start":1379,"end":1501},{"cath_id":"2.30.30.40","chopping":"1502-1597","consensus_level":"medium","plddt":84.7928,"start":1502,"end":1597}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12774","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q12774-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q12774-F1-predicted_aligned_error_v6.png","plddt_mean":47.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ARHGEF5","jax_strain_url":"https://www.jax.org/strain/search?query=ARHGEF5"},"sequence":{"accession":"Q12774","fasta_url":"https://rest.uniprot.org/uniprotkb/Q12774.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q12774/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12774"}},"corpus_meta":[{"pmid":"25363763","id":"PMC_25363763","title":"CEACAM1 regulates TIM-3-mediated tolerance and exhaustion.","date":"2014","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/25363763","citation_count":583,"is_preprint":false},{"pmid":"12776205","id":"PMC_12776205","title":"The TIM gene family: emerging roles in immunity and disease.","date":"2003","source":"Nature reviews. 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and Tim machine.","date":"1997","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/9081657","citation_count":97,"is_preprint":false},{"pmid":"28951472","id":"PMC_28951472","title":"Ebola Virus Binding to Tim-1 on T Lymphocytes Induces a Cytokine Storm.","date":"2017","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/28951472","citation_count":95,"is_preprint":false},{"pmid":"17011764","id":"PMC_17011764","title":"TIM-3 in autoimmunity.","date":"2006","source":"Current opinion in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/17011764","citation_count":90,"is_preprint":false},{"pmid":"16938542","id":"PMC_16938542","title":"TIM family of genes in immunity and tolerance.","date":"2006","source":"Advances in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/16938542","citation_count":88,"is_preprint":false},{"pmid":"25645598","id":"PMC_25645598","title":"TIM-1 signaling is required for maintenance and induction of regulatory B cells.","date":"2015","source":"American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons","url":"https://pubmed.ncbi.nlm.nih.gov/25645598","citation_count":79,"is_preprint":false},{"pmid":"33334180","id":"PMC_33334180","title":"TIM-3 pathway dysregulation and targeting in cancer.","date":"2021","source":"Expert review of anticancer therapy","url":"https://pubmed.ncbi.nlm.nih.gov/33334180","citation_count":77,"is_preprint":false},{"pmid":"29560265","id":"PMC_29560265","title":"Immune regulation by Tim-3.","date":"2018","source":"F1000Research","url":"https://pubmed.ncbi.nlm.nih.gov/29560265","citation_count":76,"is_preprint":false},{"pmid":"23555261","id":"PMC_23555261","title":"TIM-3 does not act as a receptor for galectin-9.","date":"2013","source":"PLoS 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and infection.","date":"2011","source":"Immunology letters","url":"https://pubmed.ncbi.nlm.nih.gov/21911007","citation_count":49,"is_preprint":false},{"pmid":"30876792","id":"PMC_30876792","title":"NK cell expression of Tim-3: First impressions matter.","date":"2019","source":"Immunobiology","url":"https://pubmed.ncbi.nlm.nih.gov/30876792","citation_count":47,"is_preprint":false},{"pmid":"30441759","id":"PMC_30441759","title":"TIM-1 Promotes Japanese Encephalitis Virus Entry and Infection.","date":"2018","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/30441759","citation_count":46,"is_preprint":false},{"pmid":"22294262","id":"PMC_22294262","title":"Impaired expression of Tim-3 on Th17 and Th1 cells in psoriasis.","date":"2012","source":"Acta dermato-venereologica","url":"https://pubmed.ncbi.nlm.nih.gov/22294262","citation_count":46,"is_preprint":false},{"pmid":"23143694","id":"PMC_23143694","title":"Combined blockade of TIM-3 and TIM-4 augments cancer vaccine efficacy against established melanomas.","date":"2012","source":"Cancer immunology, immunotherapy : CII","url":"https://pubmed.ncbi.nlm.nih.gov/23143694","citation_count":46,"is_preprint":false},{"pmid":"31263038","id":"PMC_31263038","title":"Tim-4 Inhibits NLRP3 Inflammasome via the LKB1/AMPKα Pathway in Macrophages.","date":"2019","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/31263038","citation_count":43,"is_preprint":false},{"pmid":"36325060","id":"PMC_36325060","title":"Cancer cell intrinsic TIM-3 induces glioblastoma progression.","date":"2022","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/36325060","citation_count":42,"is_preprint":false},{"pmid":"19713215","id":"PMC_19713215","title":"Regulation of immature dendritic cell migration by RhoA guanine nucleotide exchange factor Arhgef5.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19713215","citation_count":42,"is_preprint":false},{"pmid":"32300343","id":"PMC_32300343","title":"Tim-4 in Health and Disease: Friend or Foe?","date":"2020","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/32300343","citation_count":41,"is_preprint":false},{"pmid":"34435619","id":"PMC_34435619","title":"Phosphatidylserine binding directly regulates TIM-3 function.","date":"2021","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/34435619","citation_count":41,"is_preprint":false},{"pmid":"31242184","id":"PMC_31242184","title":"TIM-1 serves as a receptor for Ebola virus in vivo, enhancing viremia and pathogenesis.","date":"2019","source":"PLoS neglected tropical diseases","url":"https://pubmed.ncbi.nlm.nih.gov/31242184","citation_count":41,"is_preprint":false},{"pmid":"16951560","id":"PMC_16951560","title":"A Timeless debate: resolving TIM's noncircadian roles with possible clock function.","date":"2006","source":"Neuroreport","url":"https://pubmed.ncbi.nlm.nih.gov/16951560","citation_count":40,"is_preprint":false},{"pmid":"33453500","id":"PMC_33453500","title":"Evolution, folding, and design of TIM barrels and related proteins.","date":"2021","source":"Current opinion in structural biology","url":"https://pubmed.ncbi.nlm.nih.gov/33453500","citation_count":39,"is_preprint":false},{"pmid":"21906254","id":"PMC_21906254","title":"The emerging role of the TIM molecules in transplantation.","date":"2011","source":"American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons","url":"https://pubmed.ncbi.nlm.nih.gov/21906254","citation_count":38,"is_preprint":false},{"pmid":"25092608","id":"PMC_25092608","title":"Reflections on the catalytic power of a TIM-barrel.","date":"2014","source":"Bioorganic 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peptidases in bacterial physiology and host interactions.","date":"2022","source":"Cell chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/36417916","citation_count":30,"is_preprint":false},{"pmid":"27413764","id":"PMC_27413764","title":"Immune Regulation and Antitumor Effect of TIM-1.","date":"2016","source":"Journal of immunology research","url":"https://pubmed.ncbi.nlm.nih.gov/27413764","citation_count":29,"is_preprint":false},{"pmid":"19155484","id":"PMC_19155484","title":"Tim-1 signaling substitutes for conventional signal 1 and requires costimulation to induce T cell proliferation.","date":"2009","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/19155484","citation_count":29,"is_preprint":false},{"pmid":"35226805","id":"PMC_35226805","title":"Photophosphatidylserine Guides Natural Killer Cell Photoimmunotherapy via Tim-3.","date":"2022","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/35226805","citation_count":28,"is_preprint":false},{"pmid":"27617642","id":"PMC_27617642","title":"The Rho guanine nucleotide exchange factor ARHGEF5 promotes tumor malignancy via epithelial-mesenchymal transition.","date":"2016","source":"Oncogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/27617642","citation_count":28,"is_preprint":false},{"pmid":"32222966","id":"PMC_32222966","title":"TIM-3 and CEACAM1 do not interact in cis and in trans.","date":"2020","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/32222966","citation_count":27,"is_preprint":false},{"pmid":"20028372","id":"PMC_20028372","title":"Specific immunotherapy suppresses Th2 responses via modulating TIM1/TIM4 interaction on dendritic cells.","date":"2009","source":"Allergy","url":"https://pubmed.ncbi.nlm.nih.gov/20028372","citation_count":27,"is_preprint":false},{"pmid":"20232767","id":"PMC_20232767","title":"Association between TIM-1 gene polymorphisms and allergic rhinitis in a Han Chinese population.","date":"2010","source":"Journal of investigational allergology & clinical immunology","url":"https://pubmed.ncbi.nlm.nih.gov/20232767","citation_count":26,"is_preprint":false},{"pmid":"28949386","id":"PMC_28949386","title":"TIM4-TIM1 interaction modulates Th2 pattern inflammation through enhancing SIRT1 expression.","date":"2017","source":"International journal of molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/28949386","citation_count":25,"is_preprint":false},{"pmid":"31581681","id":"PMC_31581681","title":"TIM-1 As a Signal Receptor Triggers Dengue Virus-Induced Autophagy.","date":"2019","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31581681","citation_count":25,"is_preprint":false},{"pmid":"22621175","id":"PMC_22621175","title":"Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection.","date":"2012","source":"Veterinary research","url":"https://pubmed.ncbi.nlm.nih.gov/22621175","citation_count":25,"is_preprint":false},{"pmid":"32640697","id":"PMC_32640697","title":"Mertk Interacts with Tim-4 to Enhance Tim-4-Mediated Efferocytosis.","date":"2020","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/32640697","citation_count":24,"is_preprint":false},{"pmid":"30451988","id":"PMC_30451988","title":"A scaffold for signaling of Tim-4-mediated efferocytosis is formed by fibronectin.","date":"2018","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/30451988","citation_count":24,"is_preprint":false},{"pmid":"33889713","id":"PMC_33889713","title":"ImmunoPET Imaging of TIM-3 in Murine Melanoma Models.","date":"2020","source":"Advanced therapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/33889713","citation_count":23,"is_preprint":false},{"pmid":"34603337","id":"PMC_34603337","title":"Novel Roles of the Tim Family in Immune Regulation and Autoimmune Diseases.","date":"2021","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/34603337","citation_count":22,"is_preprint":false},{"pmid":"34110282","id":"PMC_34110282","title":"Ubiquitination and degradation of NF90 by Tim-3 inhibits antiviral innate immunity.","date":"2021","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/34110282","citation_count":22,"is_preprint":false},{"pmid":"28132803","id":"PMC_28132803","title":"Combined blockade of Tim-3 and MEK inhibitor enhances the efficacy against melanoma.","date":"2017","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/28132803","citation_count":21,"is_preprint":false},{"pmid":"29551917","id":"PMC_29551917","title":"On the significance of Tim-3 expression in pancreatic cancer.","date":"2017","source":"Saudi journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/29551917","citation_count":21,"is_preprint":false},{"pmid":"17161870","id":"PMC_17161870","title":"TIM-1 regulates macrophage cytokine production and B7 family member expression.","date":"2006","source":"Immunology letters","url":"https://pubmed.ncbi.nlm.nih.gov/17161870","citation_count":21,"is_preprint":false},{"pmid":"35216164","id":"PMC_35216164","title":"Association of Tim-3/Gal-9 Axis with NLRC4 Inflammasome in Glioma Malignancy: Tim-3/Gal-9 Induce the NLRC4 Inflammasome.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35216164","citation_count":21,"is_preprint":false},{"pmid":"19480659","id":"PMC_19480659","title":"Altered expression of T cell immunoglobulin-mucin (TIM) molecules in bronchoalveolar lavage CD4+ T cells in sarcoidosis.","date":"2009","source":"Respiratory research","url":"https://pubmed.ncbi.nlm.nih.gov/19480659","citation_count":21,"is_preprint":false},{"pmid":"32703939","id":"PMC_32703939","title":"Tim-4 functions as a scavenger receptor for phagocytosis of exogenous particles.","date":"2020","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/32703939","citation_count":20,"is_preprint":false},{"pmid":"31346089","id":"PMC_31346089","title":"Frustration and folding of a TIM barrel protein.","date":"2019","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/31346089","citation_count":19,"is_preprint":false},{"pmid":"23945562","id":"PMC_23945562","title":"Folding and biogenesis of mitochondrial small Tim proteins.","date":"2013","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/23945562","citation_count":19,"is_preprint":false},{"pmid":"25645980","id":"PMC_25645980","title":"The auto-inhibitory state of Rho guanine nucleotide exchange factor ARHGEF5/TIM can be relieved by targeting its SH3 domain with rationally designed peptide aptamers.","date":"2015","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/25645980","citation_count":19,"is_preprint":false},{"pmid":"26792807","id":"PMC_26792807","title":"Essential Roles of TIM-1 and TIM-4 Homologs in Adaptive Humoral Immunity in a Zebrafish Model.","date":"2016","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/26792807","citation_count":19,"is_preprint":false},{"pmid":"38916965","id":"PMC_38916965","title":"Oncogene-induced TIM-3 ligand expression dictates susceptibility to anti-TIM-3 therapy in mice.","date":"2024","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/38916965","citation_count":18,"is_preprint":false},{"pmid":"37833515","id":"PMC_37833515","title":"The phosphatidylserine receptor TIM1 promotes infection of enveloped hepatitis E virus.","date":"2023","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/37833515","citation_count":18,"is_preprint":false},{"pmid":"31438964","id":"PMC_31438964","title":"Inhibition of T cell immunoglobulin and mucin-1 (TIM-1) protects against cerebral ischemia-reperfusion injury.","date":"2019","source":"Cell communication and signaling : CCS","url":"https://pubmed.ncbi.nlm.nih.gov/31438964","citation_count":18,"is_preprint":false},{"pmid":"28357631","id":"PMC_28357631","title":"Role of TIM-3 in ovarian cancer.","date":"2017","source":"Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico","url":"https://pubmed.ncbi.nlm.nih.gov/28357631","citation_count":18,"is_preprint":false},{"pmid":"31963385","id":"PMC_31963385","title":"RhoA-GTPase Modulates Neurite Outgrowth by Regulating the Expression of Spastin and p60-Katanin.","date":"2020","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/31963385","citation_count":18,"is_preprint":false},{"pmid":"18727494","id":"PMC_18727494","title":"TIM gene family and their role in atopic diseases.","date":"2008","source":"Current topics in microbiology and immunology","url":"https://pubmed.ncbi.nlm.nih.gov/18727494","citation_count":17,"is_preprint":false},{"pmid":"18294362","id":"PMC_18294362","title":"Significant association between TIM1 promoter polymorphisms and protection against cerebral malaria in Thailand.","date":"2008","source":"Annals of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/18294362","citation_count":17,"is_preprint":false},{"pmid":"21367814","id":"PMC_21367814","title":"Polymorphisms of the TIM-1 and TIM-3 genes are not associated with systemic lupus erythematosus in a Chinese population.","date":"2011","source":"Mutagenesis","url":"https://pubmed.ncbi.nlm.nih.gov/21367814","citation_count":17,"is_preprint":false},{"pmid":"31216697","id":"PMC_31216697","title":"The Cell Wall Hydrolytic NlpC/P60 Endopeptidases in Mycobacterial Cytokinesis: A Structural Perspective.","date":"2019","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/31216697","citation_count":16,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":44421,"output_tokens":1762,"usd":0.079847},"stage2":{"model":"claude-opus-4-6","input_tokens":4759,"output_tokens":1852,"usd":0.105142},"total_usd":0.184989,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard"},"stage1_raw":"Looking through the abstracts, I need to identify papers specifically about ARHGEF5 (also known as TIM in some literature as a Rho GEF, distinct from the TIM immune family). The relevant papers are:\n\n- PMID 21525037: Kuroiwa et al., \"The guanine nucleotide exchange factor Arhgef5 plays crucial roles in Src-induced podosome formation\"\n- PMID 27617642: Komiya et al., \"The Rho guanine nucleotide exchange factor ARHGEF5 promotes tumor malignancy via epithelial-mesenchymal transition\"\n- PMID 19713215: Wang et al., \"Regulation of immature dendritic cell migration by RhoA guanine nucleotide exchange factor Arhgef5\"\n- PMID 25645980: He et al., \"The auto-inhibitory state of Rho guanine nucleotide exchange factor ARHGEF5/TIM can be relieved by targeting its SH3 domain with rationally designed peptide aptamers\"\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"ARHGEF5 (a Dbl-family Rho-GEF) was identified as a Src SH3-domain binding protein required for Src-induced podosome formation. RNAi depletion of ARHGEF5 robustly inhibited podosome formation. ARHGEF5 activates RhoA and Cdc42, is tyrosine-phosphorylated by Src, positively regulates Src kinase activity, and its PH domain is required for podosome formation. ARHGEF5 also forms a ternary complex with Src and PI3K upon Src/ARHGEF5 upregulation.\",\n      \"method\": \"Co-immunoprecipitation (Src SH3 pulldown), RNAi knockdown with podosome formation assay, overexpression with RhoA/Cdc42 activation assays, tyrosine phosphorylation assay, domain deletion/mutation analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (pulldown, RNAi phenotype, kinase assay, domain mutants) in a single study with clear mechanistic readouts\",\n      \"pmids\": [\"21525037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ARHGEF5 strongly activates RhoA and RhoB, and weakly RhoC and RhoG, but not Rac1, RhoQ, RhoD, or RhoV, as determined in HEK293 transfection assays. Gβγ subunits interact with ARHGEF5 and stimulate ARHGEF5-mediated RhoA activation in vitro. In vivo, ARHGEF5 deficiency (knockout mice) selectively abrogated MIP1α-induced chemotaxis of immature dendritic cells and impaired DC migration from skin to lymph node, while chemotaxis of macrophages, T/B lymphocytes, and mature DCs was unaffected.\",\n      \"method\": \"In vitro RhoGTPase activation assay (transfection-based), Gβγ co-immunoprecipitation, in vitro GEF assay, Arhgef5-null mouse with DC chemotaxis and skin-to-lymph-node migration assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vitro GEF assay defining substrate specificity, Gβγ interaction, and clean KO mouse with specific cellular phenotype (immature DC chemotaxis)\",\n      \"pmids\": [\"19713215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ARHGEF5 is upregulated during TGF-β-induced epithelial-mesenchymal transition (EMT) in MCF10A cells and promotes cell migration via the Rho-ROCK pathway. ARHGEF5 is required for in vitro invasion and in vivo metastasis of HCT116 colorectal cancer cells. In mesenchymal-like cells, ARHGEF5 activates Akt via the PI3K pathway to promote tumor growth, a dependence not seen in epithelial-like cells. TNF-α or Slug-induced EMT in HCT116 cells rendered tumor growth dependent on ARHGEF5.\",\n      \"method\": \"siRNA knockdown, overexpression, in vitro invasion assays, in vivo xenograft/metastasis assay, Akt/PI3K pathway analysis, EMT induction with TGF-β/TNF-α/Slug\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO/KD with specific cellular and in vivo phenotypes, pathway placement via Rho-ROCK and PI3K-Akt, single lab\",\n      \"pmids\": [\"27617642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ARHGEF5 (short isoform also called TIM) contains an auto-inhibitory mechanism in which a putative helix N-terminal to the DH domain is stabilized by intramolecular interaction between the C-terminal SH3 domain and a poly-proline sequence between the helix and the DH domain. Designed peptide aptamers that competitively bind the SH3 domain relieve auto-inhibition and activate TIM-catalyzed RhoA guanine nucleotide exchange in vitro. Mutation of Pro49, Pro52, or Lys54 in the designed peptide abolished SH3 binding and GEF activation.\",\n      \"method\": \"Molecular dynamics simulation, in vitro GEF (guanine nucleotide exchange) assay, peptide binding assay, site-directed mutagenesis of peptide aptamers\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro GEF assay with mutagenesis validating auto-inhibitory mechanism; single lab, computational model supported by biochemical validation\",\n      \"pmids\": [\"25645980\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ARHGEF5 is a Dbl-family Rho guanine nucleotide exchange factor that preferentially activates RhoA/RhoB, is held in an auto-inhibited state via an intramolecular SH3–polyproline interaction, is relieved of inhibition and activated by Src-mediated tyrosine phosphorylation and Gβγ binding, and drives Src-induced podosome formation, immature dendritic cell chemotaxis, and EMT-associated tumor invasion/metastasis through the Rho-ROCK and PI3K-Akt pathways.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ARHGEF5 is a Dbl-family Rho guanine nucleotide exchange factor that preferentially catalyzes nucleotide exchange on RhoA and RhoB, with weak activity toward RhoC and RhoG, and is held in an auto-inhibited state by an intramolecular interaction between its C-terminal SH3 domain and an internal polyproline motif that stabilizes an inhibitory helix N-terminal to the DH domain [PMID:25645980, PMID:19713215]. Relief of auto-inhibition occurs through Src-mediated tyrosine phosphorylation and Gβγ binding, enabling ARHGEF5 to drive Src-induced podosome formation via a ternary complex with Src and PI3K, and to support MIP1α-directed chemotaxis of immature dendritic cells as demonstrated by selective loss of this migration in Arhgef5-knockout mice [PMID:21525037, PMID:19713215]. ARHGEF5 is upregulated during TGF-β-induced epithelial–mesenchymal transition and promotes tumor cell invasion and metastasis through the Rho-ROCK and PI3K-Akt pathways, with mesenchymal-like cancer cells becoming selectively dependent on ARHGEF5 for Akt activation and tumor growth [PMID:27617642].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Defining substrate specificity and in vivo function: ARHGEF5 was shown to be a RhoA/RhoB-selective GEF stimulated by Gβγ subunits, and Arhgef5-null mice revealed a non-redundant requirement for immature dendritic cell chemotaxis and skin-to-lymph-node migration, establishing a specific physiological role for this exchange factor.\",\n      \"evidence\": \"In vitro GEF assays with panel of Rho GTPases, Gβγ co-immunoprecipitation, and Arhgef5-knockout mouse DC chemotaxis and migration assays\",\n      \"pmids\": [\"19713215\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism by which Gβγ relieves auto-inhibition is not structurally resolved\",\n        \"Whether ARHGEF5 functions in mature DC or other leukocyte migration under different stimuli is untested\",\n        \"No crystal structure of the ARHGEF5-RhoA catalytic complex\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Linking ARHGEF5 to Src signaling and podosomes: ARHGEF5 was identified as a Src SH3-domain partner that is tyrosine-phosphorylated by Src, positively regulates Src activity, and is required for Src-induced podosome formation through its PH domain and a ternary complex with Src and PI3K.\",\n      \"evidence\": \"Src SH3 pulldown, RNAi knockdown with podosome quantification, tyrosine phosphorylation assays, domain deletion/mutation analysis\",\n      \"pmids\": [\"21525037\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of the specific tyrosine residue(s) phosphorylated by Src is not mapped\",\n        \"How positive feedback between ARHGEF5 and Src kinase activity is regulated to avoid runaway signaling is unclear\",\n        \"Relative contributions of RhoA versus Cdc42 activation downstream of ARHGEF5 in podosome assembly are not dissected\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Elucidating the auto-inhibitory mechanism: an intramolecular SH3–polyproline interaction was shown to stabilize an inhibitory helix N-terminal to the DH domain, and competitive disruption of this interaction by designed peptide aptamers activated RhoA exchange in vitro, providing a molecular basis for regulation of ARHGEF5 catalytic activity.\",\n      \"evidence\": \"Molecular dynamics simulation, in vitro GEF assay with peptide aptamers, site-directed mutagenesis of peptide residues\",\n      \"pmids\": [\"25645980\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No experimental structure (crystal or cryo-EM) of the auto-inhibited full-length protein\",\n        \"Whether Src phosphorylation or Gβγ binding directly disrupts the SH3–polyproline interaction in cells is not demonstrated\",\n        \"In vivo validation of the peptide-aptamer activation approach is lacking\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Establishing a pro-metastatic role in EMT: ARHGEF5 was found to be upregulated during TGF-β-induced EMT and to be required for invasion and in vivo metastasis of colorectal cancer cells, operating through Rho-ROCK and PI3K-Akt signaling axes with mesenchymal-state selectivity.\",\n      \"evidence\": \"siRNA knockdown and overexpression in MCF10A and HCT116 cells, in vitro invasion assays, in vivo xenograft/metastasis models, EMT induction with TGF-β/TNF-α/Slug\",\n      \"pmids\": [\"27617642\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Transcriptional mechanism of ARHGEF5 upregulation during EMT is not defined\",\n        \"Whether ARHGEF5 dependence generalizes beyond HCT116 and MCF10A models is untested\",\n        \"Direct versus indirect activation of PI3K-Akt by ARHGEF5 in mesenchymal cells is unresolved\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full structural understanding of how Src phosphorylation, Gβγ binding, and the intramolecular SH3–polyproline switch converge to control ARHGEF5 activation remains unresolved, and additional in vivo roles beyond DC chemotaxis and cancer metastasis have not been explored.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No high-resolution structure of full-length ARHGEF5 in auto-inhibited or active states\",\n        \"Phosphosite-specific resolution of Src-mediated activation is missing\",\n        \"Potential roles in other Gβγ-coupled immune or developmental processes are unexplored\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"SRC\",\n      \"PIK3CA\",\n      \"GNB1\",\n      \"RHOA\",\n      \"RHOB\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}