{"gene":"RADIL","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2007,"finding":"RADIL was identified as a novel effector of the small GTPase Rap, required for cell adhesion and migration. Knockdown of radil in zebrafish caused defects in neural crest-derived lineages (cartilage, pigment cells, enteric neurons) primarily due to diminished migratory capacity of neural crest cells, establishing RADIL as a Rap-pathway regulator of neural crest migration.","method":"Affinity interaction assay (Rap binding), morpholino knockdown in zebrafish with phenotypic analysis of neural crest migration","journal":"Genes & development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding to Rap GTPase established, loss-of-function in vivo with specific cellular phenotype, single lab","pmids":["17704304"],"is_preprint":false},{"year":2010,"finding":"Gβγ subunits of heterotrimeric G proteins form a protein complex with activated Rap1a and Radil. This complex is required downstream of receptor stimulation for inside-out integrin activation and positive modulation of cell-matrix adhesiveness. Gβγ and activated Rap1a promote translocation of Radil to the plasma membrane at sites of cell-matrix contacts.","method":"Co-immunoprecipitation, plasma membrane translocation assay, integrin activation assay, RNAi knockdown","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and functional assays, single lab, multiple orthogonal methods","pmids":["20048162"],"is_preprint":false},{"year":2012,"finding":"Radil regulates neutrophil adhesion and motility by controlling β1- and β2-integrin activation downstream of Rap1a. On neutrophil activation, Radil translocates from cytoplasm to the plasma membrane in a Rap1a-GTP-dependent manner. Overexpression increases cell adhesion and FAK activation; knockdown inhibits β2-integrin activation and chemotaxis. Inhibition of Rap activity by RapGAP abolishes Radil-mediated integrin and FAK activation.","method":"Overexpression and RNAi knockdown in neutrophil-like cells, integrin/FAK activation assays, live-cell imaging of Radil translocation, β2-integrin inhibitory antibody rescue","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (translocation, integrin activation, FAK assay, antibody rescue, RapGAP epistasis), single lab with thorough mechanistic dissection","pmids":["23097489"],"is_preprint":false},{"year":2012,"finding":"KIF14 associates with the PDZ domain of Radil and negatively regulates Rap1-mediated inside-out integrin activation by tethering Radil on microtubules. Depletion of KIF14 leads to increased cell spreading, altered focal adhesion dynamics, and inhibition of cell migration and invasion. Radil is important for breast cancer cell proliferation and metastasis in mice.","method":"Co-immunoprecipitation (PDZ domain interaction), KIF14 knockdown with cell spreading/adhesion/migration assays, in vivo mouse metastasis model","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-specific binding established, multiple functional readouts, in vivo validation, single lab with multiple orthogonal methods","pmids":["23209302"],"is_preprint":false},{"year":2013,"finding":"Rasip1 cooperates with its close relative Radil to inhibit Rho-mediated stress fiber formation and induce junctional tightening in endothelial cells. The Rap1-Rasip1 complex induces cell spreading by inhibiting Rho signaling through ArhGAP29, and Radil participates in the Rap1-mediated regulation of endothelial junctions controlling barrier function.","method":"FRET (active Rap1 interaction), siRNA knockdown, cell spreading assay, endothelial barrier function measurement","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FRET-based interaction, functional knockdown with specific cellular readouts, single lab","pmids":["23798437"],"is_preprint":false},{"year":2013,"finding":"Protogenin (Prtg) interacts with Radil (identified by yeast two-hybrid). Overexpression of Prtg induces translocation of Radil from cytoplasm to cell membrane. Prtg and Radil together activate α5β1-integrins to high-affinity conformational forms; RNAi knockdown of Radil abolishes the effect of ERdj3/Prtg on α5β1-integrin activation, placing Radil downstream of Prtg in inside-out integrin activation in neural crest cells.","method":"Yeast two-hybrid, overexpression/translocation assay, RNAi knockdown, integrin activation assay, transwell migration assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus functional validation by RNAi epistasis, multiple methods, single lab","pmids":["23744351"],"is_preprint":false},{"year":2015,"finding":"Rap1 induces independent translocations of Rasip1 and a Radil-ArhGAP29 complex to the plasma membrane, resulting in formation of a multimeric protein complex required for Rap1-induced inhibition of Rho signaling and increased endothelial barrier function.","method":"Live-cell imaging of protein translocation, Co-immunoprecipitation, endothelial barrier function assay, siRNA knockdown","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct translocation imaging, Co-IP of complex, functional barrier assay, multiple orthogonal methods, single lab","pmids":["25963656"],"is_preprint":false},{"year":2017,"finding":"Constitutively active Gαi1(Q204L) reverses Radil-driven increases in cell spreading and adhesion to fibronectin, placing Gαi-GTP action downstream of Rap1a and Radil but upstream of integrins and talin. This negative regulation is specific to the Radil pathway, as Gαi1(Q204L) did not reverse RIAM-dependent adhesion.","method":"Expression of constitutively active mutants (Rap1a G12V, Radil, Gαi1 Q204L), cell spreading/adhesion assays, epistasis analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with defined mutants, pathway placement, single lab with multiple cell lines","pmids":["29259127"],"is_preprint":false},{"year":2018,"finding":"AF6 forms a complex with FGD5 and Radil (by co-immunoprecipitation in HEK293T cells) and mediates Rap1-enhanced tension in circumferential actin cables via Cdc42, representing a Rho-independent pathway for endothelial barrier function that operates in parallel to the Radil/Rasip1/ArhGAP29 Rho-inhibitory complex.","method":"Co-immunoprecipitation, siRNA knockdown, endothelial barrier electrical resistance measurement","journal":"Small GTPases","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP of complex plus functional barrier assay, single lab, Radil role inferred from complex membership","pmids":["29388865"],"is_preprint":false},{"year":2021,"finding":"Radil interacts with all three Ras isoforms (HRas, NRas, KRas) via its Ras-association domain, with strongest interaction for KRas; interaction is preferential for GTP-bound Ras. Ectopic Radil expression transiently activates MEK and ERK; Radil knockdown weakens Ras downstream signaling, decreases cell proliferation and invasion, reduces mesenchymal markers, and decreases adhesion foci and actin filaments.","method":"Affinity pulldown with mass spectrometry, Co-immunoprecipitation, kinase activation assay (MEK/ERK), RNAi knockdown with proliferation/invasion/adhesion assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — affinity pulldown MS plus functional Co-IP plus multiple downstream assays, single lab","pmids":["33482197"],"is_preprint":false},{"year":2021,"finding":"The PDZ domain of RADIL binds the SARS-CoV-2 E protein C-terminal PDZ-binding motif with measurable affinity (dissociation constant in the micromolar range), identified in a high-throughput screen of the full human PDZome; RADIL binding is specific to SARS-CoV-2 E and not shared with SARS-CoV E.","method":"High-throughput affinity profiling against full human PDZome (quantitative binding assay with KD measurement)","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative binding assay with KD values across full PDZome, single study, no functional follow-up for RADIL specifically","pmids":["33864728"],"is_preprint":false},{"year":2011,"finding":"In a siRNA screen, only Radil (among tested Rap effectors) blocked Rap1-induced cell spreading in A549 lung epithelial cells, establishing Radil as a required effector specifically for the cell spreading (but not basal adhesion) response downstream of Rap1.","method":"siRNA knockdown screen, Rap1-induced cell spreading assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — targeted siRNA screen with specific phenotypic readout, single lab, multiple effectors tested for specificity","pmids":["21540295"],"is_preprint":false},{"year":2005,"finding":"KIAA1849 (RADIL) contains a Ras association domain and interacted with Rap1 (GTP-bound form) but, unlike AF6, did not inhibit Rap1-induced cell adhesion when overexpressed in T cells.","method":"Co-immunoprecipitation, overexpression cell adhesion assay","journal":"The Journal of biological chemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single pulldown/overexpression experiment, negative functional result for cell adhesion inhibition, single lab","pmids":["16051602"],"is_preprint":false}],"current_model":"RADIL (also known as KIAA1849/RASIP2) is a Ras-association (RA) domain- and PDZ domain-containing adaptor protein that functions as an effector of active (GTP-bound) Rap1 and Ras GTPases: upon Rap1 activation, RADIL translocates from the cytoplasm to the plasma membrane (facilitated by Gβγ), where it forms a multimeric complex with Rasip1 and the RhoGAP ArhGAP29 to inhibit Rho/stress-fiber signaling and promote inside-out activation of β1/β2-integrins, thereby controlling cell spreading, adhesion, migration (including neural crest migration), and endothelial barrier function; its activity is negatively regulated by KIF14, which tethers RADIL on microtubules via its PDZ domain, and by Gαi-GTP acting downstream of RADIL; additionally, RADIL interacts with all three Ras isoforms (preferentially KRas-GTP) and can activate MEK-ERK signaling."},"narrative":{"mechanistic_narrative":"RADIL is a Ras-association (RA)- and PDZ-domain adaptor protein that functions as an effector of GTP-bound Rap1, coupling GTPase activation to integrin-dependent cell adhesion, spreading, and migration [PMID:17704304, PMID:23097489, PMID:21540295]. Upon receptor-driven Rap1a activation, RADIL assembles with Gβγ subunits and active Rap1a and translocates from the cytoplasm to sites of cell-matrix contact at the plasma membrane, where it drives inside-out activation of β1- and β2-integrins and downstream FAK activation [PMID:20048162, PMID:23097489]. In neural crest and other cells this pathway acts downstream of upstream cues including Protogenin to confer high-affinity integrin conformations and migratory capacity [PMID:17704304, PMID:23744351]. At endothelial junctions RADIL cooperates with its relative Rasip1 and recruits the RhoGAP ArhGAP29 into a multimeric plasma-membrane complex that suppresses Rho-mediated stress fiber formation and promotes barrier function [PMID:23798437, PMID:25963656]. RADIL activity is negatively regulated at two levels: KIF14 tethers RADIL on microtubules through its PDZ domain to restrain Rap1-mediated integrin activation, and Gαi-GTP acts downstream of Rap1a/RADIL but upstream of integrins to reverse RADIL-driven adhesion [PMID:23209302, PMID:29259127]. RADIL also binds all three Ras isoforms via its RA domain, preferentially GTP-bound KRas, and supports Ras-MEK-ERK signaling, proliferation, and invasion, consistent with a role in breast cancer metastasis [PMID:23209302, PMID:33482197].","teleology":[{"year":2005,"claim":"Established that the uncharacterized KIAA1849 protein bears a Ras-association domain and physically engages GTP-bound Rap1, raising the question of whether it is a functional Rap effector.","evidence":"Co-immunoprecipitation and overexpression adhesion assay in T cells","pmids":["16051602"],"confidence":"Low","gaps":["Single pulldown with a negative functional adhesion result","Effector function and cellular role not demonstrated","No domain-mapping of the binding interface"]},{"year":2007,"claim":"Defined RADIL as a genuine Rap effector required for cell adhesion and migration, and linked it in vivo to neural crest migration, establishing its developmental and cellular relevance.","evidence":"Rap-binding affinity assay plus morpholino knockdown in zebrafish with neural crest phenotyping","pmids":["17704304"],"confidence":"Medium","gaps":["Molecular mechanism connecting RADIL to migration not resolved","Single lab","Downstream effectors at the membrane not identified"]},{"year":2010,"claim":"Showed how RADIL is brought to its site of action by demonstrating that Gβγ and activated Rap1a form a complex with RADIL that drives plasma-membrane translocation and inside-out integrin activation downstream of receptor stimulation.","evidence":"Reciprocal Co-IP, membrane translocation assay, integrin activation assay, RNAi","pmids":["20048162"],"confidence":"Medium","gaps":["Structural basis of the Gβγ–Rap1a–RADIL complex unknown","How the complex activates integrins mechanistically not defined"]},{"year":2011,"claim":"Resolved which Rap effector mediates spreading by showing RADIL is uniquely required, among tested effectors, for Rap1-induced cell spreading but not basal adhesion, establishing pathway specificity.","evidence":"siRNA effector screen with Rap1-induced spreading assay in A549 cells","pmids":["21540295"],"confidence":"Medium","gaps":["Distinction between spreading and adhesion machinery not mechanistically explained","Single cell type"]},{"year":2012,"claim":"Provided thorough mechanistic dissection in neutrophils, placing RADIL downstream of Rap1a-GTP for translocation and showing it controls β1/β2-integrin and FAK activation, chemotaxis, and adhesion.","evidence":"Overexpression/RNAi, live-cell translocation imaging, integrin/FAK assays, RapGAP epistasis, integrin antibody rescue","pmids":["23097489"],"confidence":"High","gaps":["Direct biochemical step linking RADIL to talin/integrin not isolated","In vivo neutrophil function not tested"]},{"year":2012,"claim":"Identified a negative regulator: KIF14 binds the RADIL PDZ domain and sequesters it on microtubules to restrain Rap1-mediated integrin activation, and tied RADIL to breast cancer proliferation and metastasis.","evidence":"PDZ-domain Co-IP, KIF14 knockdown spreading/adhesion/migration assays, in vivo mouse metastasis model","pmids":["23209302"],"confidence":"High","gaps":["How microtubule release of RADIL is triggered unknown","Mechanism by which RADIL promotes metastasis not fully defined"]},{"year":2013,"claim":"Extended RADIL function to endothelial biology, showing it cooperates with Rasip1 to inhibit Rho-mediated stress fibers, promote spreading via ArhGAP29, and regulate junctional barrier function.","evidence":"FRET interaction, siRNA, spreading and endothelial barrier assays","pmids":["23798437"],"confidence":"Medium","gaps":["Stoichiometry and order of Rasip1/RADIL/ArhGAP29 assembly unresolved at this stage","Direct ArhGAP29 recruitment by RADIL not yet shown"]},{"year":2013,"claim":"Placed RADIL downstream of Protogenin in inside-out integrin activation, identifying an upstream receptor input that drives RADIL membrane translocation and α5β1-integrin activation in neural crest cells.","evidence":"Yeast two-hybrid, translocation assay, RNAi epistasis, integrin activation and transwell migration assays","pmids":["23744351"],"confidence":"Medium","gaps":["Direct vs indirect nature of Prtg–RADIL coupling at the membrane unclear","Generality beyond neural crest cells not established"]},{"year":2015,"claim":"Clarified complex assembly by showing Rap1 independently recruits Rasip1 and a RADIL-ArhGAP29 complex to the membrane, forming a multimeric assembly that inhibits Rho signaling and strengthens the endothelial barrier.","evidence":"Live-cell translocation imaging, Co-IP, endothelial barrier assay, siRNA","pmids":["25963656"],"confidence":"High","gaps":["Structural architecture of the multimeric complex unknown","Dynamics of complex turnover not characterized"]},{"year":2017,"claim":"Positioned a second negative regulator, showing Gαi-GTP acts downstream of Rap1a/RADIL but upstream of integrins/talin to reverse RADIL-driven adhesion, with specificity for the RADIL (not RIAM) pathway.","evidence":"Constitutively active mutant epistasis (Rap1a G12V, RADIL, Gαi1 Q204L), spreading/adhesion assays","pmids":["29259127"],"confidence":"Medium","gaps":["Biochemical target of Gαi-GTP within the pathway not identified","Mechanism of RADIL-pathway specificity unexplained"]},{"year":2018,"claim":"Defined a parallel Rho-independent endothelial barrier route in which AF6/FGD5/RADIL drives Cdc42-mediated actin tension, distinguishing it from the RADIL/Rasip1/ArhGAP29 Rho-inhibitory arm.","evidence":"Co-IP, siRNA, endothelial barrier electrical resistance measurement","pmids":["29388865"],"confidence":"Medium","gaps":["RADIL's specific contribution inferred from complex membership rather than direct assay","Relationship between the two parallel pathways not resolved"]},{"year":2021,"claim":"Broadened RADIL's GTPase repertoire beyond Rap, showing RA-domain binding to GTP-bound Ras isoforms (preferentially KRas) and a role in MEK-ERK signaling, proliferation, invasion, and mesenchymal phenotype.","evidence":"Affinity pulldown with mass spectrometry, Co-IP, MEK/ERK kinase assay, RNAi with proliferation/invasion/adhesion assays","pmids":["33482197"],"confidence":"Medium","gaps":["Whether Ras and Rap binding are mutually exclusive unknown","How RADIL feeds into the MEK-ERK cascade mechanistically not defined"]},{"year":2021,"claim":"Identified the RADIL PDZ domain as a micromolar binder of the SARS-CoV-2 E protein PDZ-binding motif, with specificity over SARS-CoV E, flagging a potential viral hijacking interaction.","evidence":"High-throughput quantitative affinity profiling against the full human PDZome with KD measurement","pmids":["33864728"],"confidence":"Medium","gaps":["No functional consequence for RADIL demonstrated","Interaction not validated in infected cells"]},{"year":null,"claim":"How RADIL integrates competing inputs — Rap versus Ras GTPase binding, positive membrane recruitment versus KIF14/Gαi negative regulation — into a single decision controlling adhesion, spreading, and barrier function remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of RADIL or its multimeric complexes","Switch between Rap-adhesion and Ras-ERK functions not defined","Physiological signals controlling KIF14 release and Gαi action unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,4,7]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,2,6]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,2]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[4,6]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,5]}],"complexes":["Rap1a-Gβγ-RADIL complex","RADIL-Rasip1-ArhGAP29 complex","AF6-FGD5-RADIL complex"],"partners":["RAP1A","KIF14","RASIP1","ARHGAP29","PRTG","KRAS","MLLT4","FGD5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96JH8","full_name":"Ras-associating and dilute domain-containing protein","aliases":[],"length_aa":1075,"mass_kda":117.5,"function":"Downstream effector of Rap required for cell adhesion and migration of neural crest precursors during development","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q96JH8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RADIL","classification":"Not Classified","n_dependent_lines":12,"n_total_lines":1208,"dependency_fraction":0.009933774834437087},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RADIL","total_profiled":1310},"omim":[{"mim_id":"611491","title":"RAS ASSOCIATION AND DILUTE DOMAINS PROTEIN; RADIL","url":"https://www.omim.org/entry/611491"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Nuclear membrane","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":8.4}],"url":"https://www.proteinatlas.org/search/RADIL"},"hgnc":{"alias_symbol":["FLJ10324","KIAA1849","RASIP2"],"prev_symbol":[]},"alphafold":{"accession":"Q96JH8","domains":[{"cath_id":"3.10.20.90","chopping":"52-173","consensus_level":"high","plddt":82.9226,"start":52,"end":173},{"cath_id":"2.60.200.20","chopping":"246-306_313-373","consensus_level":"high","plddt":83.5989,"start":246,"end":373},{"cath_id":"-","chopping":"630-762","consensus_level":"high","plddt":83.8768,"start":630,"end":762},{"cath_id":"2.30.42.10","chopping":"975-1069","consensus_level":"high","plddt":87.2376,"start":975,"end":1069},{"cath_id":"1.20.1310","chopping":"413-571","consensus_level":"high","plddt":84.8048,"start":413,"end":571}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96JH8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96JH8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96JH8-F1-predicted_aligned_error_v6.png","plddt_mean":68.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RADIL","jax_strain_url":"https://www.jax.org/strain/search?query=RADIL"},"sequence":{"accession":"Q96JH8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96JH8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96JH8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96JH8"}},"corpus_meta":[{"pmid":"23798437","id":"PMC_23798437","title":"Rasip1 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Survivors of Anaplastic Thyroid Cancer: A Genomic Predictive Model.","date":"2026","source":"The Journal of clinical endocrinology and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/40663630","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14625,"output_tokens":3353,"usd":0.047085,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10900,"output_tokens":4248,"usd":0.08035,"stage2_stop_reason":"end_turn"},"total_usd":0.127435,"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\": 2007,\n      \"finding\": \"RADIL was identified as a novel effector of the small GTPase Rap, required for cell adhesion and migration. Knockdown of radil in zebrafish caused defects in neural crest-derived lineages (cartilage, pigment cells, enteric neurons) primarily due to diminished migratory capacity of neural crest cells, establishing RADIL as a Rap-pathway regulator of neural crest migration.\",\n      \"method\": \"Affinity interaction assay (Rap binding), morpholino knockdown in zebrafish with phenotypic analysis of neural crest migration\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding to Rap GTPase established, loss-of-function in vivo with specific cellular phenotype, single lab\",\n      \"pmids\": [\"17704304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Gβγ subunits of heterotrimeric G proteins form a protein complex with activated Rap1a and Radil. This complex is required downstream of receptor stimulation for inside-out integrin activation and positive modulation of cell-matrix adhesiveness. Gβγ and activated Rap1a promote translocation of Radil to the plasma membrane at sites of cell-matrix contacts.\",\n      \"method\": \"Co-immunoprecipitation, plasma membrane translocation assay, integrin activation assay, RNAi knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and functional assays, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"20048162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Radil regulates neutrophil adhesion and motility by controlling β1- and β2-integrin activation downstream of Rap1a. On neutrophil activation, Radil translocates from cytoplasm to the plasma membrane in a Rap1a-GTP-dependent manner. Overexpression increases cell adhesion and FAK activation; knockdown inhibits β2-integrin activation and chemotaxis. Inhibition of Rap activity by RapGAP abolishes Radil-mediated integrin and FAK activation.\",\n      \"method\": \"Overexpression and RNAi knockdown in neutrophil-like cells, integrin/FAK activation assays, live-cell imaging of Radil translocation, β2-integrin inhibitory antibody rescue\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (translocation, integrin activation, FAK assay, antibody rescue, RapGAP epistasis), single lab with thorough mechanistic dissection\",\n      \"pmids\": [\"23097489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"KIF14 associates with the PDZ domain of Radil and negatively regulates Rap1-mediated inside-out integrin activation by tethering Radil on microtubules. Depletion of KIF14 leads to increased cell spreading, altered focal adhesion dynamics, and inhibition of cell migration and invasion. Radil is important for breast cancer cell proliferation and metastasis in mice.\",\n      \"method\": \"Co-immunoprecipitation (PDZ domain interaction), KIF14 knockdown with cell spreading/adhesion/migration assays, in vivo mouse metastasis model\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-specific binding established, multiple functional readouts, in vivo validation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"23209302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Rasip1 cooperates with its close relative Radil to inhibit Rho-mediated stress fiber formation and induce junctional tightening in endothelial cells. The Rap1-Rasip1 complex induces cell spreading by inhibiting Rho signaling through ArhGAP29, and Radil participates in the Rap1-mediated regulation of endothelial junctions controlling barrier function.\",\n      \"method\": \"FRET (active Rap1 interaction), siRNA knockdown, cell spreading assay, endothelial barrier function measurement\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRET-based interaction, functional knockdown with specific cellular readouts, single lab\",\n      \"pmids\": [\"23798437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Protogenin (Prtg) interacts with Radil (identified by yeast two-hybrid). Overexpression of Prtg induces translocation of Radil from cytoplasm to cell membrane. Prtg and Radil together activate α5β1-integrins to high-affinity conformational forms; RNAi knockdown of Radil abolishes the effect of ERdj3/Prtg on α5β1-integrin activation, placing Radil downstream of Prtg in inside-out integrin activation in neural crest cells.\",\n      \"method\": \"Yeast two-hybrid, overexpression/translocation assay, RNAi knockdown, integrin activation assay, transwell migration assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus functional validation by RNAi epistasis, multiple methods, single lab\",\n      \"pmids\": [\"23744351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Rap1 induces independent translocations of Rasip1 and a Radil-ArhGAP29 complex to the plasma membrane, resulting in formation of a multimeric protein complex required for Rap1-induced inhibition of Rho signaling and increased endothelial barrier function.\",\n      \"method\": \"Live-cell imaging of protein translocation, Co-immunoprecipitation, endothelial barrier function assay, siRNA knockdown\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct translocation imaging, Co-IP of complex, functional barrier assay, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"25963656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Constitutively active Gαi1(Q204L) reverses Radil-driven increases in cell spreading and adhesion to fibronectin, placing Gαi-GTP action downstream of Rap1a and Radil but upstream of integrins and talin. This negative regulation is specific to the Radil pathway, as Gαi1(Q204L) did not reverse RIAM-dependent adhesion.\",\n      \"method\": \"Expression of constitutively active mutants (Rap1a G12V, Radil, Gαi1 Q204L), cell spreading/adhesion assays, epistasis analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with defined mutants, pathway placement, single lab with multiple cell lines\",\n      \"pmids\": [\"29259127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"AF6 forms a complex with FGD5 and Radil (by co-immunoprecipitation in HEK293T cells) and mediates Rap1-enhanced tension in circumferential actin cables via Cdc42, representing a Rho-independent pathway for endothelial barrier function that operates in parallel to the Radil/Rasip1/ArhGAP29 Rho-inhibitory complex.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, endothelial barrier electrical resistance measurement\",\n      \"journal\": \"Small GTPases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP of complex plus functional barrier assay, single lab, Radil role inferred from complex membership\",\n      \"pmids\": [\"29388865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Radil interacts with all three Ras isoforms (HRas, NRas, KRas) via its Ras-association domain, with strongest interaction for KRas; interaction is preferential for GTP-bound Ras. Ectopic Radil expression transiently activates MEK and ERK; Radil knockdown weakens Ras downstream signaling, decreases cell proliferation and invasion, reduces mesenchymal markers, and decreases adhesion foci and actin filaments.\",\n      \"method\": \"Affinity pulldown with mass spectrometry, Co-immunoprecipitation, kinase activation assay (MEK/ERK), RNAi knockdown with proliferation/invasion/adhesion assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — affinity pulldown MS plus functional Co-IP plus multiple downstream assays, single lab\",\n      \"pmids\": [\"33482197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The PDZ domain of RADIL binds the SARS-CoV-2 E protein C-terminal PDZ-binding motif with measurable affinity (dissociation constant in the micromolar range), identified in a high-throughput screen of the full human PDZome; RADIL binding is specific to SARS-CoV-2 E and not shared with SARS-CoV E.\",\n      \"method\": \"High-throughput affinity profiling against full human PDZome (quantitative binding assay with KD measurement)\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative binding assay with KD values across full PDZome, single study, no functional follow-up for RADIL specifically\",\n      \"pmids\": [\"33864728\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In a siRNA screen, only Radil (among tested Rap effectors) blocked Rap1-induced cell spreading in A549 lung epithelial cells, establishing Radil as a required effector specifically for the cell spreading (but not basal adhesion) response downstream of Rap1.\",\n      \"method\": \"siRNA knockdown screen, Rap1-induced cell spreading assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — targeted siRNA screen with specific phenotypic readout, single lab, multiple effectors tested for specificity\",\n      \"pmids\": [\"21540295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"KIAA1849 (RADIL) contains a Ras association domain and interacted with Rap1 (GTP-bound form) but, unlike AF6, did not inhibit Rap1-induced cell adhesion when overexpressed in T cells.\",\n      \"method\": \"Co-immunoprecipitation, overexpression cell adhesion assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single pulldown/overexpression experiment, negative functional result for cell adhesion inhibition, single lab\",\n      \"pmids\": [\"16051602\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RADIL (also known as KIAA1849/RASIP2) is a Ras-association (RA) domain- and PDZ domain-containing adaptor protein that functions as an effector of active (GTP-bound) Rap1 and Ras GTPases: upon Rap1 activation, RADIL translocates from the cytoplasm to the plasma membrane (facilitated by Gβγ), where it forms a multimeric complex with Rasip1 and the RhoGAP ArhGAP29 to inhibit Rho/stress-fiber signaling and promote inside-out activation of β1/β2-integrins, thereby controlling cell spreading, adhesion, migration (including neural crest migration), and endothelial barrier function; its activity is negatively regulated by KIF14, which tethers RADIL on microtubules via its PDZ domain, and by Gαi-GTP acting downstream of RADIL; additionally, RADIL interacts with all three Ras isoforms (preferentially KRas-GTP) and can activate MEK-ERK signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RADIL is a Ras-association (RA)- and PDZ-domain adaptor protein that functions as an effector of GTP-bound Rap1, coupling GTPase activation to integrin-dependent cell adhesion, spreading, and migration [#0, #2, #11]. Upon receptor-driven Rap1a activation, RADIL assembles with Gβγ subunits and active Rap1a and translocates from the cytoplasm to sites of cell-matrix contact at the plasma membrane, where it drives inside-out activation of β1- and β2-integrins and downstream FAK activation [#1, #2]. In neural crest and other cells this pathway acts downstream of upstream cues including Protogenin to confer high-affinity integrin conformations and migratory capacity [#0, #5]. At endothelial junctions RADIL cooperates with its relative Rasip1 and recruits the RhoGAP ArhGAP29 into a multimeric plasma-membrane complex that suppresses Rho-mediated stress fiber formation and promotes barrier function [#4, #6]. RADIL activity is negatively regulated at two levels: KIF14 tethers RADIL on microtubules through its PDZ domain to restrain Rap1-mediated integrin activation, and Gαi-GTP acts downstream of Rap1a/RADIL but upstream of integrins to reverse RADIL-driven adhesion [#3, #7]. RADIL also binds all three Ras isoforms via its RA domain, preferentially GTP-bound KRas, and supports Ras-MEK-ERK signaling, proliferation, and invasion, consistent with a role in breast cancer metastasis [#3, #9].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established that the uncharacterized KIAA1849 protein bears a Ras-association domain and physically engages GTP-bound Rap1, raising the question of whether it is a functional Rap effector.\",\n      \"evidence\": \"Co-immunoprecipitation and overexpression adhesion assay in T cells\",\n      \"pmids\": [\"16051602\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single pulldown with a negative functional adhesion result\", \"Effector function and cellular role not demonstrated\", \"No domain-mapping of the binding interface\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined RADIL as a genuine Rap effector required for cell adhesion and migration, and linked it in vivo to neural crest migration, establishing its developmental and cellular relevance.\",\n      \"evidence\": \"Rap-binding affinity assay plus morpholino knockdown in zebrafish with neural crest phenotyping\",\n      \"pmids\": [\"17704304\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism connecting RADIL to migration not resolved\", \"Single lab\", \"Downstream effectors at the membrane not identified\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed how RADIL is brought to its site of action by demonstrating that Gβγ and activated Rap1a form a complex with RADIL that drives plasma-membrane translocation and inside-out integrin activation downstream of receptor stimulation.\",\n      \"evidence\": \"Reciprocal Co-IP, membrane translocation assay, integrin activation assay, RNAi\",\n      \"pmids\": [\"20048162\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of the Gβγ–Rap1a–RADIL complex unknown\", \"How the complex activates integrins mechanistically not defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved which Rap effector mediates spreading by showing RADIL is uniquely required, among tested effectors, for Rap1-induced cell spreading but not basal adhesion, establishing pathway specificity.\",\n      \"evidence\": \"siRNA effector screen with Rap1-induced spreading assay in A549 cells\",\n      \"pmids\": [\"21540295\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Distinction between spreading and adhesion machinery not mechanistically explained\", \"Single cell type\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Provided thorough mechanistic dissection in neutrophils, placing RADIL downstream of Rap1a-GTP for translocation and showing it controls β1/β2-integrin and FAK activation, chemotaxis, and adhesion.\",\n      \"evidence\": \"Overexpression/RNAi, live-cell translocation imaging, integrin/FAK assays, RapGAP epistasis, integrin antibody rescue\",\n      \"pmids\": [\"23097489\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical step linking RADIL to talin/integrin not isolated\", \"In vivo neutrophil function not tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified a negative regulator: KIF14 binds the RADIL PDZ domain and sequesters it on microtubules to restrain Rap1-mediated integrin activation, and tied RADIL to breast cancer proliferation and metastasis.\",\n      \"evidence\": \"PDZ-domain Co-IP, KIF14 knockdown spreading/adhesion/migration assays, in vivo mouse metastasis model\",\n      \"pmids\": [\"23209302\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How microtubule release of RADIL is triggered unknown\", \"Mechanism by which RADIL promotes metastasis not fully defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended RADIL function to endothelial biology, showing it cooperates with Rasip1 to inhibit Rho-mediated stress fibers, promote spreading via ArhGAP29, and regulate junctional barrier function.\",\n      \"evidence\": \"FRET interaction, siRNA, spreading and endothelial barrier assays\",\n      \"pmids\": [\"23798437\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry and order of Rasip1/RADIL/ArhGAP29 assembly unresolved at this stage\", \"Direct ArhGAP29 recruitment by RADIL not yet shown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed RADIL downstream of Protogenin in inside-out integrin activation, identifying an upstream receptor input that drives RADIL membrane translocation and α5β1-integrin activation in neural crest cells.\",\n      \"evidence\": \"Yeast two-hybrid, translocation assay, RNAi epistasis, integrin activation and transwell migration assays\",\n      \"pmids\": [\"23744351\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect nature of Prtg–RADIL coupling at the membrane unclear\", \"Generality beyond neural crest cells not established\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Clarified complex assembly by showing Rap1 independently recruits Rasip1 and a RADIL-ArhGAP29 complex to the membrane, forming a multimeric assembly that inhibits Rho signaling and strengthens the endothelial barrier.\",\n      \"evidence\": \"Live-cell translocation imaging, Co-IP, endothelial barrier assay, siRNA\",\n      \"pmids\": [\"25963656\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural architecture of the multimeric complex unknown\", \"Dynamics of complex turnover not characterized\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Positioned a second negative regulator, showing Gαi-GTP acts downstream of Rap1a/RADIL but upstream of integrins/talin to reverse RADIL-driven adhesion, with specificity for the RADIL (not RIAM) pathway.\",\n      \"evidence\": \"Constitutively active mutant epistasis (Rap1a G12V, RADIL, Gαi1 Q204L), spreading/adhesion assays\",\n      \"pmids\": [\"29259127\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Biochemical target of Gαi-GTP within the pathway not identified\", \"Mechanism of RADIL-pathway specificity unexplained\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined a parallel Rho-independent endothelial barrier route in which AF6/FGD5/RADIL drives Cdc42-mediated actin tension, distinguishing it from the RADIL/Rasip1/ArhGAP29 Rho-inhibitory arm.\",\n      \"evidence\": \"Co-IP, siRNA, endothelial barrier electrical resistance measurement\",\n      \"pmids\": [\"29388865\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RADIL's specific contribution inferred from complex membership rather than direct assay\", \"Relationship between the two parallel pathways not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Broadened RADIL's GTPase repertoire beyond Rap, showing RA-domain binding to GTP-bound Ras isoforms (preferentially KRas) and a role in MEK-ERK signaling, proliferation, invasion, and mesenchymal phenotype.\",\n      \"evidence\": \"Affinity pulldown with mass spectrometry, Co-IP, MEK/ERK kinase assay, RNAi with proliferation/invasion/adhesion assays\",\n      \"pmids\": [\"33482197\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Ras and Rap binding are mutually exclusive unknown\", \"How RADIL feeds into the MEK-ERK cascade mechanistically not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified the RADIL PDZ domain as a micromolar binder of the SARS-CoV-2 E protein PDZ-binding motif, with specificity over SARS-CoV E, flagging a potential viral hijacking interaction.\",\n      \"evidence\": \"High-throughput quantitative affinity profiling against the full human PDZome with KD measurement\",\n      \"pmids\": [\"33864728\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional consequence for RADIL demonstrated\", \"Interaction not validated in infected cells\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RADIL integrates competing inputs — Rap versus Ras GTPase binding, positive membrane recruitment versus KIF14/Gαi negative regulation — into a single decision controlling adhesion, spreading, and barrier function remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of RADIL or its multimeric complexes\", \"Switch between Rap-adhesion and Ras-ERK functions not defined\", \"Physiological signals controlling KIF14 release and Gαi action unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 4, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 2, 6]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [4, 6]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"complexes\": [\n      \"Rap1a-Gβγ-RADIL complex\",\n      \"RADIL-Rasip1-ArhGAP29 complex\",\n      \"AF6-FGD5-RADIL complex\"\n    ],\n    \"partners\": [\n      \"RAP1A\",\n      \"KIF14\",\n      \"RASIP1\",\n      \"ARHGAP29\",\n      \"PRTG\",\n      \"KRAS\",\n      \"MLLT4\",\n      \"FGD5\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}