{"gene":"RAPGEF6","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2001,"finding":"RA-GEF-2 (RAPGEF6) is a guanine nucleotide exchange factor that stimulates nucleotide exchange on both Rap1 and Rap2, but not Ha-Ras. Its RA domain binds M-Ras in a GTP-dependent manner (but not other Ras family GTPases tested, including Ha-Ras, N-Ras, Rap1A, Rap2A, R-Ras, RalA, Rin, Rit, or Rheb). Upon coexpression with activated M-Ras, RA-GEF-2 colocalizes with it at the plasma membrane and increases GTP-bound Rap1 there, in contrast to the related RA-GEF-1, which acts in perinuclear/Golgi compartments.","method":"In vitro GEF assay, domain-specific binding assays, subcellular colocalization in COS-7 cells, coexpression experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro GEF activity assay plus domain-binding specificity experiments plus subcellular colocalization, multiple orthogonal methods in one study","pmids":["11524421"],"is_preprint":false},{"year":2007,"finding":"The M-Ras → RA-GEF-2 → Rap1 pathway mediates TNF-α-triggered LFA-1 integrin activation in hematopoietic/splenocyte cells. Activated M-Ras induces LFA-1-mediated cell aggregation that is abrogated by knockdown of RA-GEF-2 or Rap1. TNF-α activates LFA-1 in a manner dependent on M-Ras, RA-GEF-2, and Rap1 and recruits RA-GEF-2 to the plasma membrane. Genetic deletion of RA-GEF-2 in mice confirmed its requirement for TNF-α-stimulated, Rap1-mediated LFA-1 activation in splenocytes.","method":"siRNA knockdown, genetic knockout mice, cell aggregation assays, Rap1 activation assays, subcellular localization experiments","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis by knockdown and genetic KO mice, multiple orthogonal methods, replicated in primary splenocytes","pmids":["17538012"],"is_preprint":false},{"year":2008,"finding":"PDZ-GEF2 (RAPGEF6) is required for maturation of adherens junctions in lung carcinoma A549 cells. Knockdown of PDZ-GEF2 results in persistence of adhesion zippers at cell-cell contacts. Constitutively active Rap1A rescues junction maturation in the absence of PDZ-GEF2, placing Rap1A downstream of PDZ-GEF2 in this process. PDZ-GEF2 knockdown also reduces E-cadherin levels (an effect mimicked by Rap1B but not Rap1A siRNA), indicating PDZ-GEF2 activates both Rap1A (junction maturation) and Rap1B (E-cadherin regulation) for distinct functions.","method":"siRNA knockdown, constitutively active Rap1A rescue, immunofluorescence microscopy, E-cadherin level measurement","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 / Moderate — epistasis established by loss-of-function plus genetic rescue with active Rap1A, two orthogonal phenotypic readouts, single lab","pmids":["18585005"],"is_preprint":false},{"year":2009,"finding":"JAM-A forms a physical complex with Afadin and PDZ-GEF2 at tight junctions in epithelial cells. This complex is functionally required: loss of JAM-A, Afadin, or PDZ-GEF2 (but not ZO-1 or PDZ-GEF1) similarly decreases active Rap1, β1 integrin protein levels, and cell migration. Association of PDZ-GEF2 with Afadin is dependent on JAM-A expression. Downstream, Rap1A (but not Rap1B) knockdown phenocopies the decrease in β1 integrin and migration, establishing JAM-A → Afadin/PDZ-GEF2 → Rap1A → β1 integrin → cell migration as the functional pathway.","method":"Co-immunoprecipitation, siRNA knockdown, cell migration assays, β1 integrin quantification","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus functional knockdown epistasis with multiple readouts, single lab","pmids":["19176753"],"is_preprint":false},{"year":2010,"finding":"BAG3 directly interacts with PDZGEF2 (RAPGEF6) via binding of the PPDY motif at the C-terminus of PDZGEF2 to the WW domain of BAG3, both in vitro and in vivo. PDZGEF2 activates Rap1 and increases integrin-mediated cell adhesion. BAG3 overexpression increases cell adhesion, but this effect is lost when PDZGEF2 is knocked down, demonstrating that PDZGEF2 is required downstream of BAG3 for cell adhesion regulation.","method":"Co-immunoprecipitation, domain deletion mutants, in vitro binding assay, siRNA knockdown, cell adhesion assay on fibronectin","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo binding with defined domain interaction, functional epistasis by knockdown, single lab","pmids":["20800573"],"is_preprint":false},{"year":2014,"finding":"RA-GEF-2/Rapgef6 is required for spermatogenesis in mice. Knockout of RA-GEF-2 results in testicular atrophy, hypospermatogenesis, reduced sperm concentration and motility, abnormal sperm morphology, and male infertility. Loss of RA-GEF-2 causes reduced expression and disrupted junctional localization of N-cadherin in testes, suggesting the Rapgef6-Rap1 pathway maintains N-cadherin-dependent cell junctions necessary for testicular differentiation.","method":"Genetic knockout mice, histological analysis, sperm parameter measurement, N-cadherin immunolocalization","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined phenotypic readouts including molecular marker (N-cadherin localization), single lab","pmids":["24491570"],"is_preprint":false},{"year":2015,"finding":"Deletion of Rapgef6 in mice impairs amygdala function, evidenced by reduced fear conditioning and anxiolysis, reduced cFOS phosphorylation in hippocampus and amygdala after fear conditioning, reduced spine density and primary dendrite number in CA3 pyramidal neurons, and enhanced long-term potentiation at cortico-amygdala synapses.","method":"Genetic knockout mice, fear conditioning behavioral assays, cFOS immunohistochemistry, dendritic morphology analysis, electrophysiology (LTP measurement)","journal":"Translational psychiatry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with multiple orthogonal neural readouts (behavior, IHC, morphology, electrophysiology), single lab","pmids":["26057047"],"is_preprint":false},{"year":2016,"finding":"Rapgef2 and Rapgef6 act redundantly and cell-autonomously through a Rap1 pathway to maintain apical surface adherens junctions in radial glial cells during mouse cerebral cortex development. Double knockout (Rapgef2/6-dKO) causes greater disruption of apical AJs, RGC detachment, and radial glial fiber disorganization than single Rapgef2 knockout alone, affecting both early-born and late-born neurons. Cotransduction of constitutively active Rap1(G12V) suppresses AJ disruption and RGC detachment caused by Rapgef2 loss, placing Rap1 downstream of Rapgef2/6 in this pathway.","method":"Conditional and double genetic knockout mice, intrauterine Cre transduction, constitutively active Rap1 rescue, immunohistochemistry for AJ markers, confocal microscopy","journal":"eNeuro","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with active Rap1 rescue in vivo, double KO strategy, multiple orthogonal methods including in utero rescue experiment","pmids":["27390776"],"is_preprint":false},{"year":2021,"finding":"Desmoglein-2 (Dsg2) controls cell spreading and focal adhesion phosphorylation through a PDZ-GEF2/Rap1 signaling axis. Cells lacking Dsg2 show elevated Rap1 activity and increased spreading on fibronectin and collagen, even in single cells (independent of cell-cell adhesion). PDZ-GEF2 was implicated as mediating Dsg2-dependent Rap1 activation, with downstream TGFβ signaling also involved.","method":"Dsg2 knockout cells, Rap1 activity assay, cell spreading area measurement, focal adhesion phosphorylation assay, siRNA for PDZ-GEF2","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO phenotype with Rap1 activity measurement and PDZ-GEF2 implicated by knockdown, single lab, multiple readouts","pmids":["34168237"],"is_preprint":false},{"year":2025,"finding":"Stable knockdown of RAPGEF6 in RAW264.7 macrophages attenuates LPS-induced inflammatory responses and oxidative stress by inhibiting NF-κB-mediated necroptosis.","method":"Lentiviral shRNA stable knockdown, LPS stimulation, NF-κB pathway assays, necroptosis readouts","journal":"Functional & integrative genomics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single knockdown model, pathway placement inferred from downstream markers without direct mechanistic reconstitution","pmids":["41081959"],"is_preprint":false}],"current_model":"RAPGEF6 (RA-GEF-2/PDZ-GEF2) is a guanine nucleotide exchange factor that activates Rap1 and Rap2 GTPases; it is recruited to the plasma membrane by binding activated M-Ras through its RA domain, where it functions downstream of upstream receptors (JAM-A, Desmoglein-2, TNF-α receptor via M-Ras) in complexes with scaffold proteins (Afadin, BAG3) to activate Rap1A and/or Rap1B, thereby regulating cell-cell junction maturation (adherens junctions), cell-matrix adhesion and spreading (β1 integrin levels, focal adhesions), cell migration, integrin-mediated leukocyte adhesion (LFA-1), spermatogenesis (via N-cadherin junctions), and neural progenitor apical adherens junction integrity during cortical development."},"narrative":{"mechanistic_narrative":"RAPGEF6 (RA-GEF-2/PDZ-GEF2) is a guanine nucleotide exchange factor that activates the Rap1 and Rap2 GTPases to control cell-cell junction maturation, cell-matrix adhesion, and cell migration [PMID:11524421, PMID:18585005, PMID:19176753]. It catalyzes nucleotide exchange on Rap1 and Rap2 but not Ha-Ras, and its RA domain binds GTP-loaded M-Ras specifically, recruiting the GEF to the plasma membrane where it locally elevates active Rap1 [PMID:11524421]. This M-Ras–RAPGEF6–Rap1 module drives TNF-α–triggered LFA-1 integrin activation in splenocytes, a requirement confirmed by knockout mice [PMID:17538012]. At epithelial junctions RAPGEF6 acts within a JAM-A–Afadin scaffold, where its association with Afadin depends on JAM-A and the resulting Rap1A activation maintains β1 integrin levels and supports cell migration, while activating Rap1A and Rap1B for distinct outputs—adherens junction maturation versus E-cadherin regulation [PMID:18585005, PMID:19176753]. The C-terminal PPDY motif of RAPGEF6 binds the WW domain of the co-chaperone BAG3, and RAPGEF6 is required downstream of BAG3 for integrin-mediated adhesion to fibronectin [PMID:20800573]. RAPGEF6 also operates downstream of desmoglein-2 to restrain Rap1 activity and cell spreading [PMID:34168237]. In vivo, RAPGEF6 acts redundantly with RAPGEF2 through Rap1 to maintain apical adherens junctions in cortical radial glia [PMID:27390776], is required for N-cadherin junction integrity during spermatogenesis [PMID:24491570], and supports amygdala-dependent fear behavior and neuronal morphology [PMID:26057047].","teleology":[{"year":2001,"claim":"Established RAPGEF6 as a Rap-specific GEF and defined how it is targeted to the plasma membrane, answering both its catalytic specificity and its upstream activator.","evidence":"In vitro GEF assays, GTP-dependent domain-binding specificity tests, and colocalization in COS-7 cells","pmids":["11524421"],"confidence":"High","gaps":["Physiological receptor inputs upstream of M-Ras not yet defined","No structural basis for RA domain–M-Ras selectivity"]},{"year":2007,"claim":"Placed RAPGEF6 in a signaling chain from a physiological stimulus to integrin function, showing M-Ras→RAPGEF6→Rap1 mediates TNF-α–induced LFA-1 activation.","evidence":"siRNA knockdown, genetic knockout mice, cell aggregation and Rap1 activation assays in splenocytes","pmids":["17538012"],"confidence":"High","gaps":["How the TNF-α receptor connects to M-Ras activation is unresolved","Direct membrane recruitment mechanism beyond M-Ras binding not dissected"]},{"year":2008,"claim":"Demonstrated that RAPGEF6 routes distinct Rap1 isoforms to distinct adhesion outputs, resolving how one GEF supports both junction maturation and cadherin regulation.","evidence":"siRNA knockdown with constitutively active Rap1A rescue and E-cadherin quantification in A549 cells","pmids":["18585005"],"confidence":"High","gaps":["Molecular basis of Rap1A versus Rap1B selectivity unknown","Single cell-line context"]},{"year":2009,"claim":"Identified the scaffold that positions RAPGEF6 at junctions, defining a JAM-A→Afadin/RAPGEF6→Rap1A→β1 integrin migration pathway.","evidence":"Reciprocal co-immunoprecipitation, siRNA knockdown epistasis, and migration/β1 integrin readouts","pmids":["19176753"],"confidence":"High","gaps":["Whether RAPGEF6 binds Afadin directly or via JAM-A not distinguished","Quantitative stoichiometry of the complex unknown"]},{"year":2010,"claim":"Mapped a direct PPDY–WW interaction linking RAPGEF6 to the co-chaperone BAG3, placing RAPGEF6 downstream of BAG3 in adhesion control.","evidence":"Co-IP, domain-deletion mutants, in vitro binding, and adhesion assays on fibronectin","pmids":["20800573"],"confidence":"Medium","gaps":["Functional consequence of BAG3 binding for GEF activity not measured","Single-lab finding"]},{"year":2014,"claim":"Extended RAPGEF6 function to a whole-organism adhesion phenotype, showing it maintains N-cadherin junctions required for spermatogenesis.","evidence":"Genetic knockout mice with histology, sperm parameters, and N-cadherin immunolocalization","pmids":["24491570"],"confidence":"Medium","gaps":["Direct link between Rap1 activity and N-cadherin junction maintenance in testes not established","Cell type responsible not pinpointed"]},{"year":2015,"claim":"Revealed a neural role for RAPGEF6 in amygdala-dependent behavior and neuronal structure.","evidence":"Knockout mice analyzed by fear conditioning, cFOS IHC, dendritic morphology, and LTP electrophysiology","pmids":["26057047"],"confidence":"Medium","gaps":["Molecular pathway connecting RAPGEF6 to synaptic phenotypes not defined","Whether effects are Rap1-dependent untested"]},{"year":2016,"claim":"Showed RAPGEF6 acts redundantly with RAPGEF2 via Rap1 to maintain apical adherens junctions in radial glia, resolving its developmental role through in vivo epistasis.","evidence":"Conditional/double knockout mice with in utero Cre transduction and constitutively active Rap1 rescue","pmids":["27390776"],"confidence":"High","gaps":["Upstream cue activating Rapgef2/6 in radial glia unknown","Basis of functional redundancy between the two GEFs not dissected"]},{"year":2021,"claim":"Placed RAPGEF6 downstream of desmoglein-2 in restraining Rap1 activity and cell spreading, extending its input repertoire to desmosomal cadherins.","evidence":"Dsg2 knockout cells with Rap1 activity assays, spreading measurement, and RAPGEF6 knockdown","pmids":["34168237"],"confidence":"Medium","gaps":["How Dsg2 loss elevates RAPGEF6/Rap1 activity mechanistically unclear","Role of the implicated TGFβ arm not integrated"]},{"year":2025,"claim":"Linked RAPGEF6 to macrophage inflammatory and necroptotic responses, a distinct functional context from its adhesion roles.","evidence":"Lentiviral shRNA stable knockdown in RAW264.7 macrophages with LPS stimulation and NF-κB/necroptosis readouts","pmids":["41081959"],"confidence":"Low","gaps":["Pathway placement inferred from downstream markers without mechanistic reconstitution","No demonstration that GEF/Rap1 activity mediates the effect","Single knockdown model"]},{"year":null,"claim":"How RAPGEF6 selects between Rap1A and Rap1B and integrates its multiple upstream inputs (M-Ras, JAM-A/Afadin, BAG3, Dsg2) into context-specific outputs remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of substrate or activator selection","Mechanistic basis for tissue-specific output unknown","Direct GEF activity not connected to neural or inflammatory phenotypes"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,8]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[2,3]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[5,7]}],"complexes":["JAM-A–Afadin–PDZ-GEF2 junctional complex"],"partners":["MRAS","F11R","MLLT4","BAG3","DSG2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8TEU7","full_name":"Rap guanine nucleotide exchange factor 6","aliases":["PDZ domain-containing guanine nucleotide exchange factor 2","PDZ-GEF2","RA-GEF-2"],"length_aa":1601,"mass_kda":179.4,"function":"Guanine nucleotide exchange factor (GEF) for Rap1A, Rap2A and M-Ras GTPases. Does not interact with cAMP","subcellular_location":"Cytoplasm; Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q8TEU7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RAPGEF6","classification":"Not Classified","n_dependent_lines":19,"n_total_lines":1208,"dependency_fraction":0.015728476821192054},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RAPGEF6","total_profiled":1310},"omim":[{"mim_id":"614531","title":"RASGEF DOMAIN FAMILY, MEMBER 1A; RASGEF1A","url":"https://www.omim.org/entry/614531"},{"mim_id":"610499","title":"RAP GUANINE NUCLEOTIDE EXCHANGE FACTOR 6; RAPGEF6","url":"https://www.omim.org/entry/610499"},{"mim_id":"301016","title":"RAS-RELATED PROTEIN 2C; RAP2C","url":"https://www.omim.org/entry/301016"},{"mim_id":"179541","title":"RAS-RELATED PROTEIN 2B; RAP2B","url":"https://www.omim.org/entry/179541"},{"mim_id":"179540","title":"RAS-RELATED PROTEIN 2A; RAP2A","url":"https://www.omim.org/entry/179540"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Centrosome","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RAPGEF6"},"hgnc":{"alias_symbol":["RA-GEF-2","PDZ-GEF2"],"prev_symbol":["PDZGEF2"]},"alphafold":{"accession":"Q8TEU7","domains":[{"cath_id":"2.60.120.10","chopping":"8-120","consensus_level":"high","plddt":81.3265,"start":8,"end":120},{"cath_id":"2.60.120.10","chopping":"250-393","consensus_level":"medium","plddt":85.0227,"start":250,"end":393},{"cath_id":"2.30.42.10","chopping":"528-611","consensus_level":"medium","plddt":87.942,"start":528,"end":611},{"cath_id":"3.10.20.90","chopping":"752-852","consensus_level":"high","plddt":87.8002,"start":752,"end":852},{"cath_id":"1.10.840.10","chopping":"856-1012_1058-1065_1099-1121","consensus_level":"high","plddt":89.2355,"start":856,"end":1121}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TEU7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TEU7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TEU7-F1-predicted_aligned_error_v6.png","plddt_mean":60.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RAPGEF6","jax_strain_url":"https://www.jax.org/strain/search?query=RAPGEF6"},"sequence":{"accession":"Q8TEU7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TEU7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TEU7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TEU7"}},"corpus_meta":[{"pmid":"19176753","id":"PMC_19176753","title":"Junctional adhesion molecule A interacts with Afadin and PDZ-GEF2 to activate Rap1A, regulate beta1 integrin levels, and enhance cell migration.","date":"2009","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/19176753","citation_count":146,"is_preprint":false},{"pmid":"11524421","id":"PMC_11524421","title":"Identification and characterization of RA-GEF-2, a Rap guanine nucleotide exchange factor that serves as a downstream target of M-Ras.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11524421","citation_count":80,"is_preprint":false},{"pmid":"20800573","id":"PMC_20800573","title":"BAG3 directly associates with guanine nucleotide exchange factor of Rap1, PDZGEF2, and regulates cell adhesion.","date":"2010","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/20800573","citation_count":61,"is_preprint":false},{"pmid":"17538012","id":"PMC_17538012","title":"The M-Ras-RA-GEF-2-Rap1 pathway mediates tumor necrosis factor-alpha dependent regulation of integrin activation in splenocytes.","date":"2007","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/17538012","citation_count":44,"is_preprint":false},{"pmid":"17030554","id":"PMC_17030554","title":"Haplotypes spanning SPEC2, PDZ-GEF2 and ACSL6 genes are associated with schizophrenia.","date":"2006","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/17030554","citation_count":43,"is_preprint":false},{"pmid":"18585005","id":"PMC_18585005","title":"The RapGEF PDZ-GEF2 is required for maturation of cell-cell junctions.","date":"2008","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/18585005","citation_count":40,"is_preprint":false},{"pmid":"27390776","id":"PMC_27390776","title":"Crucial Role of Rapgef2 and Rapgef6, a Family of Guanine Nucleotide Exchange Factors for Rap1 Small GTPase, in Formation of Apical Surface Adherens Junctions and Neural Progenitor Development in the Mouse Cerebral Cortex.","date":"2016","source":"eNeuro","url":"https://pubmed.ncbi.nlm.nih.gov/27390776","citation_count":25,"is_preprint":false},{"pmid":"18718982","id":"PMC_18718982","title":"Association of haplotypes spanning PDZ-GEF2, LOC728637 and ACSL6 with schizophrenia in Han Chinese.","date":"2008","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/18718982","citation_count":21,"is_preprint":false},{"pmid":"26057047","id":"PMC_26057047","title":"Deletion of Rapgef6, a candidate schizophrenia susceptibility gene, disrupts amygdala function in mice.","date":"2015","source":"Translational psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/26057047","citation_count":18,"is_preprint":false},{"pmid":"24491570","id":"PMC_24491570","title":"Critical function of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in mouse spermatogenesis.","date":"2014","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/24491570","citation_count":13,"is_preprint":false},{"pmid":"31137587","id":"PMC_31137587","title":"Novel Polymorphisms in RAPGEF6 Gene Associated with Egg-Laying Rate in Chinese Jing Hong Chicken using Genome-Wide SNP Scan.","date":"2019","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/31137587","citation_count":11,"is_preprint":false},{"pmid":"34168237","id":"PMC_34168237","title":"Desmoglein-2 harnesses a PDZ-GEF2/Rap1 signaling axis to control cell spreading and focal adhesions independent of cell-cell adhesion.","date":"2021","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/34168237","citation_count":10,"is_preprint":false},{"pmid":"31647804","id":"PMC_31647804","title":"A RAPGEF6 variant constitutes a major risk factor for laryngeal paralysis in dogs.","date":"2019","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31647804","citation_count":7,"is_preprint":false},{"pmid":"39923209","id":"PMC_39923209","title":"Novel ETV6::RAPGEF6 fusion gene in chronic eosinophilic leukemia: compiling evidence on the role of IL3 overexpression in tumorigenesis.","date":"2025","source":"Annals of hematology","url":"https://pubmed.ncbi.nlm.nih.gov/39923209","citation_count":2,"is_preprint":false},{"pmid":"41081959","id":"PMC_41081959","title":"RAPGEF6 is a key biomarker with temporal characteristics in sepsis and exacerbates inflammation and oxidative stress by NF-κB-mediated necroptosis.","date":"2025","source":"Functional & integrative genomics","url":"https://pubmed.ncbi.nlm.nih.gov/41081959","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10582,"output_tokens":3178,"usd":0.039708,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10609,"output_tokens":3467,"usd":0.06986,"stage2_stop_reason":"end_turn"},"total_usd":0.109568,"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\": 2001,\n      \"finding\": \"RA-GEF-2 (RAPGEF6) is a guanine nucleotide exchange factor that stimulates nucleotide exchange on both Rap1 and Rap2, but not Ha-Ras. Its RA domain binds M-Ras in a GTP-dependent manner (but not other Ras family GTPases tested, including Ha-Ras, N-Ras, Rap1A, Rap2A, R-Ras, RalA, Rin, Rit, or Rheb). Upon coexpression with activated M-Ras, RA-GEF-2 colocalizes with it at the plasma membrane and increases GTP-bound Rap1 there, in contrast to the related RA-GEF-1, which acts in perinuclear/Golgi compartments.\",\n      \"method\": \"In vitro GEF assay, domain-specific binding assays, subcellular colocalization in COS-7 cells, coexpression experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro GEF activity assay plus domain-binding specificity experiments plus subcellular colocalization, multiple orthogonal methods in one study\",\n      \"pmids\": [\"11524421\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The M-Ras → RA-GEF-2 → Rap1 pathway mediates TNF-α-triggered LFA-1 integrin activation in hematopoietic/splenocyte cells. Activated M-Ras induces LFA-1-mediated cell aggregation that is abrogated by knockdown of RA-GEF-2 or Rap1. TNF-α activates LFA-1 in a manner dependent on M-Ras, RA-GEF-2, and Rap1 and recruits RA-GEF-2 to the plasma membrane. Genetic deletion of RA-GEF-2 in mice confirmed its requirement for TNF-α-stimulated, Rap1-mediated LFA-1 activation in splenocytes.\",\n      \"method\": \"siRNA knockdown, genetic knockout mice, cell aggregation assays, Rap1 activation assays, subcellular localization experiments\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis by knockdown and genetic KO mice, multiple orthogonal methods, replicated in primary splenocytes\",\n      \"pmids\": [\"17538012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PDZ-GEF2 (RAPGEF6) is required for maturation of adherens junctions in lung carcinoma A549 cells. Knockdown of PDZ-GEF2 results in persistence of adhesion zippers at cell-cell contacts. Constitutively active Rap1A rescues junction maturation in the absence of PDZ-GEF2, placing Rap1A downstream of PDZ-GEF2 in this process. PDZ-GEF2 knockdown also reduces E-cadherin levels (an effect mimicked by Rap1B but not Rap1A siRNA), indicating PDZ-GEF2 activates both Rap1A (junction maturation) and Rap1B (E-cadherin regulation) for distinct functions.\",\n      \"method\": \"siRNA knockdown, constitutively active Rap1A rescue, immunofluorescence microscopy, E-cadherin level measurement\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis established by loss-of-function plus genetic rescue with active Rap1A, two orthogonal phenotypic readouts, single lab\",\n      \"pmids\": [\"18585005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"JAM-A forms a physical complex with Afadin and PDZ-GEF2 at tight junctions in epithelial cells. This complex is functionally required: loss of JAM-A, Afadin, or PDZ-GEF2 (but not ZO-1 or PDZ-GEF1) similarly decreases active Rap1, β1 integrin protein levels, and cell migration. Association of PDZ-GEF2 with Afadin is dependent on JAM-A expression. Downstream, Rap1A (but not Rap1B) knockdown phenocopies the decrease in β1 integrin and migration, establishing JAM-A → Afadin/PDZ-GEF2 → Rap1A → β1 integrin → cell migration as the functional pathway.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, cell migration assays, β1 integrin quantification\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus functional knockdown epistasis with multiple readouts, single lab\",\n      \"pmids\": [\"19176753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"BAG3 directly interacts with PDZGEF2 (RAPGEF6) via binding of the PPDY motif at the C-terminus of PDZGEF2 to the WW domain of BAG3, both in vitro and in vivo. PDZGEF2 activates Rap1 and increases integrin-mediated cell adhesion. BAG3 overexpression increases cell adhesion, but this effect is lost when PDZGEF2 is knocked down, demonstrating that PDZGEF2 is required downstream of BAG3 for cell adhesion regulation.\",\n      \"method\": \"Co-immunoprecipitation, domain deletion mutants, in vitro binding assay, siRNA knockdown, cell adhesion assay on fibronectin\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo binding with defined domain interaction, functional epistasis by knockdown, single lab\",\n      \"pmids\": [\"20800573\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RA-GEF-2/Rapgef6 is required for spermatogenesis in mice. Knockout of RA-GEF-2 results in testicular atrophy, hypospermatogenesis, reduced sperm concentration and motility, abnormal sperm morphology, and male infertility. Loss of RA-GEF-2 causes reduced expression and disrupted junctional localization of N-cadherin in testes, suggesting the Rapgef6-Rap1 pathway maintains N-cadherin-dependent cell junctions necessary for testicular differentiation.\",\n      \"method\": \"Genetic knockout mice, histological analysis, sperm parameter measurement, N-cadherin immunolocalization\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined phenotypic readouts including molecular marker (N-cadherin localization), single lab\",\n      \"pmids\": [\"24491570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Deletion of Rapgef6 in mice impairs amygdala function, evidenced by reduced fear conditioning and anxiolysis, reduced cFOS phosphorylation in hippocampus and amygdala after fear conditioning, reduced spine density and primary dendrite number in CA3 pyramidal neurons, and enhanced long-term potentiation at cortico-amygdala synapses.\",\n      \"method\": \"Genetic knockout mice, fear conditioning behavioral assays, cFOS immunohistochemistry, dendritic morphology analysis, electrophysiology (LTP measurement)\",\n      \"journal\": \"Translational psychiatry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with multiple orthogonal neural readouts (behavior, IHC, morphology, electrophysiology), single lab\",\n      \"pmids\": [\"26057047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Rapgef2 and Rapgef6 act redundantly and cell-autonomously through a Rap1 pathway to maintain apical surface adherens junctions in radial glial cells during mouse cerebral cortex development. Double knockout (Rapgef2/6-dKO) causes greater disruption of apical AJs, RGC detachment, and radial glial fiber disorganization than single Rapgef2 knockout alone, affecting both early-born and late-born neurons. Cotransduction of constitutively active Rap1(G12V) suppresses AJ disruption and RGC detachment caused by Rapgef2 loss, placing Rap1 downstream of Rapgef2/6 in this pathway.\",\n      \"method\": \"Conditional and double genetic knockout mice, intrauterine Cre transduction, constitutively active Rap1 rescue, immunohistochemistry for AJ markers, confocal microscopy\",\n      \"journal\": \"eNeuro\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with active Rap1 rescue in vivo, double KO strategy, multiple orthogonal methods including in utero rescue experiment\",\n      \"pmids\": [\"27390776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Desmoglein-2 (Dsg2) controls cell spreading and focal adhesion phosphorylation through a PDZ-GEF2/Rap1 signaling axis. Cells lacking Dsg2 show elevated Rap1 activity and increased spreading on fibronectin and collagen, even in single cells (independent of cell-cell adhesion). PDZ-GEF2 was implicated as mediating Dsg2-dependent Rap1 activation, with downstream TGFβ signaling also involved.\",\n      \"method\": \"Dsg2 knockout cells, Rap1 activity assay, cell spreading area measurement, focal adhesion phosphorylation assay, siRNA for PDZ-GEF2\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO phenotype with Rap1 activity measurement and PDZ-GEF2 implicated by knockdown, single lab, multiple readouts\",\n      \"pmids\": [\"34168237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Stable knockdown of RAPGEF6 in RAW264.7 macrophages attenuates LPS-induced inflammatory responses and oxidative stress by inhibiting NF-κB-mediated necroptosis.\",\n      \"method\": \"Lentiviral shRNA stable knockdown, LPS stimulation, NF-κB pathway assays, necroptosis readouts\",\n      \"journal\": \"Functional & integrative genomics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single knockdown model, pathway placement inferred from downstream markers without direct mechanistic reconstitution\",\n      \"pmids\": [\"41081959\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RAPGEF6 (RA-GEF-2/PDZ-GEF2) is a guanine nucleotide exchange factor that activates Rap1 and Rap2 GTPases; it is recruited to the plasma membrane by binding activated M-Ras through its RA domain, where it functions downstream of upstream receptors (JAM-A, Desmoglein-2, TNF-α receptor via M-Ras) in complexes with scaffold proteins (Afadin, BAG3) to activate Rap1A and/or Rap1B, thereby regulating cell-cell junction maturation (adherens junctions), cell-matrix adhesion and spreading (β1 integrin levels, focal adhesions), cell migration, integrin-mediated leukocyte adhesion (LFA-1), spermatogenesis (via N-cadherin junctions), and neural progenitor apical adherens junction integrity during cortical development.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RAPGEF6 (RA-GEF-2/PDZ-GEF2) is a guanine nucleotide exchange factor that activates the Rap1 and Rap2 GTPases to control cell-cell junction maturation, cell-matrix adhesion, and cell migration [#0, #2, #3]. It catalyzes nucleotide exchange on Rap1 and Rap2 but not Ha-Ras, and its RA domain binds GTP-loaded M-Ras specifically, recruiting the GEF to the plasma membrane where it locally elevates active Rap1 [#0]. This M-Ras–RAPGEF6–Rap1 module drives TNF-\\u03b1–triggered LFA-1 integrin activation in splenocytes, a requirement confirmed by knockout mice [#1]. At epithelial junctions RAPGEF6 acts within a JAM-A–Afadin scaffold, where its association with Afadin depends on JAM-A and the resulting Rap1A activation maintains \\u03b21 integrin levels and supports cell migration, while activating Rap1A and Rap1B for distinct outputs—adherens junction maturation versus E-cadherin regulation [#2, #3]. The C-terminal PPDY motif of RAPGEF6 binds the WW domain of the co-chaperone BAG3, and RAPGEF6 is required downstream of BAG3 for integrin-mediated adhesion to fibronectin [#4]. RAPGEF6 also operates downstream of desmoglein-2 to restrain Rap1 activity and cell spreading [#8]. In vivo, RAPGEF6 acts redundantly with RAPGEF2 through Rap1 to maintain apical adherens junctions in cortical radial glia [#7], is required for N-cadherin junction integrity during spermatogenesis [#5], and supports amygdala-dependent fear behavior and neuronal morphology [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established RAPGEF6 as a Rap-specific GEF and defined how it is targeted to the plasma membrane, answering both its catalytic specificity and its upstream activator.\",\n      \"evidence\": \"In vitro GEF assays, GTP-dependent domain-binding specificity tests, and colocalization in COS-7 cells\",\n      \"pmids\": [\"11524421\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological receptor inputs upstream of M-Ras not yet defined\", \"No structural basis for RA domain–M-Ras selectivity\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Placed RAPGEF6 in a signaling chain from a physiological stimulus to integrin function, showing M-Ras\\u2192RAPGEF6\\u2192Rap1 mediates TNF-\\u03b1\\u2013induced LFA-1 activation.\",\n      \"evidence\": \"siRNA knockdown, genetic knockout mice, cell aggregation and Rap1 activation assays in splenocytes\",\n      \"pmids\": [\"17538012\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the TNF-\\u03b1 receptor connects to M-Ras activation is unresolved\", \"Direct membrane recruitment mechanism beyond M-Ras binding not dissected\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated that RAPGEF6 routes distinct Rap1 isoforms to distinct adhesion outputs, resolving how one GEF supports both junction maturation and cadherin regulation.\",\n      \"evidence\": \"siRNA knockdown with constitutively active Rap1A rescue and E-cadherin quantification in A549 cells\",\n      \"pmids\": [\"18585005\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of Rap1A versus Rap1B selectivity unknown\", \"Single cell-line context\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified the scaffold that positions RAPGEF6 at junctions, defining a JAM-A\\u2192Afadin/RAPGEF6\\u2192Rap1A\\u2192\\u03b21 integrin migration pathway.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, siRNA knockdown epistasis, and migration/\\u03b21 integrin readouts\",\n      \"pmids\": [\"19176753\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RAPGEF6 binds Afadin directly or via JAM-A not distinguished\", \"Quantitative stoichiometry of the complex unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mapped a direct PPDY–WW interaction linking RAPGEF6 to the co-chaperone BAG3, placing RAPGEF6 downstream of BAG3 in adhesion control.\",\n      \"evidence\": \"Co-IP, domain-deletion mutants, in vitro binding, and adhesion assays on fibronectin\",\n      \"pmids\": [\"20800573\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of BAG3 binding for GEF activity not measured\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended RAPGEF6 function to a whole-organism adhesion phenotype, showing it maintains N-cadherin junctions required for spermatogenesis.\",\n      \"evidence\": \"Genetic knockout mice with histology, sperm parameters, and N-cadherin immunolocalization\",\n      \"pmids\": [\"24491570\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct link between Rap1 activity and N-cadherin junction maintenance in testes not established\", \"Cell type responsible not pinpointed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revealed a neural role for RAPGEF6 in amygdala-dependent behavior and neuronal structure.\",\n      \"evidence\": \"Knockout mice analyzed by fear conditioning, cFOS IHC, dendritic morphology, and LTP electrophysiology\",\n      \"pmids\": [\"26057047\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular pathway connecting RAPGEF6 to synaptic phenotypes not defined\", \"Whether effects are Rap1-dependent untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed RAPGEF6 acts redundantly with RAPGEF2 via Rap1 to maintain apical adherens junctions in radial glia, resolving its developmental role through in vivo epistasis.\",\n      \"evidence\": \"Conditional/double knockout mice with in utero Cre transduction and constitutively active Rap1 rescue\",\n      \"pmids\": [\"27390776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream cue activating Rapgef2/6 in radial glia unknown\", \"Basis of functional redundancy between the two GEFs not dissected\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed RAPGEF6 downstream of desmoglein-2 in restraining Rap1 activity and cell spreading, extending its input repertoire to desmosomal cadherins.\",\n      \"evidence\": \"Dsg2 knockout cells with Rap1 activity assays, spreading measurement, and RAPGEF6 knockdown\",\n      \"pmids\": [\"34168237\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How Dsg2 loss elevates RAPGEF6/Rap1 activity mechanistically unclear\", \"Role of the implicated TGF\\u03b2 arm not integrated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked RAPGEF6 to macrophage inflammatory and necroptotic responses, a distinct functional context from its adhesion roles.\",\n      \"evidence\": \"Lentiviral shRNA stable knockdown in RAW264.7 macrophages with LPS stimulation and NF-\\u03baB/necroptosis readouts\",\n      \"pmids\": [\"41081959\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Pathway placement inferred from downstream markers without mechanistic reconstitution\", \"No demonstration that GEF/Rap1 activity mediates the effect\", \"Single knockdown model\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RAPGEF6 selects between Rap1A and Rap1B and integrates its multiple upstream inputs (M-Ras, JAM-A/Afadin, BAG3, Dsg2) into context-specific outputs remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of substrate or activator selection\", \"Mechanistic basis for tissue-specific output unknown\", \"Direct GEF activity not connected to neural or inflammatory phenotypes\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3]},\n      {\"term_id\": \"GO:0005085\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 8]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"complexes\": [\"JAM-A–Afadin–PDZ-GEF2 junctional complex\"],\n    \"partners\": [\"MRAS\", \"F11R\", \"MLLT4\", \"BAG3\", \"DSG2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}