{"gene":"LAD1","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":1996,"finding":"LAD-1 (Ladinin-1) is a novel 120-kDa anchoring filament protein synthesized and secreted by epidermal keratinocytes, localizing exclusively to anchoring filaments at the dermal-epidermal junction as determined by immunoelectron microscopy. A monoclonal antibody against LAD-1 induced dermal-epidermal separation of human skin in situ, establishing its role in maintaining dermal-epidermal cohesion.","method":"Radioimmunoprecipitation, immunoelectron microscopy, immunoblot, in situ antibody-induced blistering assay, primary keratinocyte culture","journal":"The Journal of investigative dermatology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (immunoelectron microscopy, radioimmunoprecipitation, functional in situ blistering assay) in a single rigorous characterization study","pmids":["8618013"],"is_preprint":false},{"year":1999,"finding":"LAD-1 exists as a high-molecular-mass homotrimeric complex (analogous to BP180 trimers) in human skin and keratinocytes, and does not form mixed complexes with BP180, as shown by two-dimensional electrophoresis under temperature-dissociation conditions.","method":"Two-dimensional SDS-PAGE with temperature-controlled dissociation, immunoblotting, silver staining","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal methods (2D gel + immunoblot) in a single lab study definitively showing homotrimeric complex and absence of BP180 mixed complexes","pmids":["9886264"],"is_preprint":false},{"year":1997,"finding":"LAD-1 protein expression is absent or greatly reduced in the majority of generalized atrophic benign epidermolysis bullosa (GABEB) patients, and this defect co-occurs with abnormal BP180 expression, as confirmed by indirect immunofluorescence and radioimmunoprecipitation from cultured keratinocytes.","method":"Indirect immunofluorescence microscopy, radioimmunoprecipitation from keratinocyte cultures","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal methods (immunofluorescence + radioimmunoprecipitation) in a single lab with a cohort of 44 patients","pmids":["9284104"],"is_preprint":false},{"year":2010,"finding":"LAD1 (Ladinin-1) functions as an adaptor protein in the EGF-to-ERK5 signaling pathway: EGF stimulation increases MEKK2 binding to LAD1 in a calcium-dependent manner, and this direct interaction (demonstrated by in vitro binding assay) is required for MEKK2 nuclear translocation and full tyrosine phosphorylation of MEKK2, thereby enabling ERK5 activation.","method":"Co-immunoprecipitation, in vitro binding assay, intracellular calcium modifiers, nuclear translocation imaging","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP plus in vitro direct binding assay in a single lab with multiple orthogonal approaches","pmids":["20830310"],"is_preprint":false},{"year":2018,"finding":"LAD1 (Ladinin-1) is transcriptionally induced by EGF, phosphorylated downstream of EGF signaling, and binds to actin-crosslinking filamin proteins; it co-localizes with actin stress fibers and participates in actin dynamics in collaboration with 14-3-3σ (SFN), mediating mammary cell proliferation and migration.","method":"Phosphoproteomics (SILAC), yeast two-hybrid, proximity ligation assay, co-immunoprecipitation, cosedimentation assay, shRNA knockdown, xenograft tumor model","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (SILAC phosphoproteomics, yeast two-hybrid, PLA, co-IP, cosedimentation, in vivo xenograft) in a single rigorous study","pmids":["29382783"],"is_preprint":false},{"year":2023,"finding":"LAD1 promotes gastric cancer progression by competitively binding to Vimentin, preventing Vimentin's interaction with the E3 ubiquitin ligase MAEA, thereby reducing K48-linked ubiquitination and degradation of Vimentin, leading to increased Vimentin protein levels.","method":"Mass spectrometry (interactome), co-immunoprecipitation, ubiquitination assay, in vitro and in vivo functional gain/loss experiments (PDX and CDX xenograft models)","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, MS-based interactome identification, and ubiquitination assay in a single lab with in vivo validation","pmids":["37718450"],"is_preprint":false},{"year":2025,"finding":"LAD1 (Ladinin-1) co-regulates cortactin (CTTN) and N-WASP phosphorylation to mediate cytoskeletal reorganization in esophageal squamous cell carcinoma (ESCC), and together with LINC01305 promotes EMT via activation of the PI3K/AKT signaling pathway.","method":"Co-IP (LAD1-LINC01305 binding), phosphorylation assays for CTTN and N-WASP, EMT marker analysis, PI3K/AKT pathway assays, functional migration/invasion assays","journal":"Molecular carcinogenesis","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, mechanistic details limited in abstract; binding to a lncRNA and downstream phosphorylation readouts without full reconstitution","pmids":["39835575"],"is_preprint":false},{"year":2020,"finding":"LAD1 depletion inhibits migration and invasion of colorectal cancer cells (SW620, Caco-2) and reduces liver metastasis in a spleen-to-liver mouse model, without affecting cell proliferation, indicating a specific role in metastatic behavior.","method":"shRNA-mediated LAD1 depletion, migration/invasion assays, in vivo spleen-to-liver metastasis mouse model, immunohistochemistry","journal":"BMC cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined loss-of-function phenotype with both in vitro and in vivo readouts in a single lab","pmids":["33267790"],"is_preprint":false}],"current_model":"LAD1 (Ladinin-1) is a homotrimeric anchoring filament protein secreted by keratinocytes that maintains dermal-epidermal cohesion; it also acts as an intracellular signaling adaptor that binds MEKK2 in a calcium-dependent manner to relay EGF signals to ERK5, binds filamin proteins to regulate actin dynamics and cell migration downstream of EGF, and in cancer contexts protects Vimentin from MAEA-mediated ubiquitin-dependent degradation, collectively promoting cell migration, invasion, and metastasis."},"narrative":{"mechanistic_narrative":"LAD1 (Ladinin-1) is a 120-kDa anchoring filament protein originally characterized as a keratinocyte-secreted component of the dermal-epidermal junction, where antibody-induced disruption causes dermal-epidermal separation, establishing its role in maintaining dermal-epidermal cohesion [PMID:8618013]. In skin it assembles as a homotrimeric complex distinct from BP180 [PMID:9886264], and its loss accompanies abnormal BP180 expression in generalized atrophic benign epidermolysis bullosa patients [PMID:9284104]. Beyond its structural role, LAD1 acts as an intracellular signaling adaptor downstream of EGF: it binds MEKK2 in a calcium-dependent manner to enable MEKK2 nuclear translocation, tyrosine phosphorylation, and ERK5 activation [PMID:20830310], and is itself transcriptionally induced and phosphorylated by EGF signaling, binding actin-crosslinking filamin proteins and co-localizing with actin stress fibers together with 14-3-3σ to drive cell proliferation and migration [PMID:29382783]. In cancer, LAD1 promotes migratory and metastatic behavior — its depletion blocks colorectal cancer migration, invasion, and liver metastasis without affecting proliferation [PMID:33267790] — and it stabilizes Vimentin by competitively blocking the MAEA E3 ligase, reducing K48-linked ubiquitination and degradation of Vimentin in gastric cancer [PMID:37718450].","teleology":[{"year":1996,"claim":"Establishing whether LAD1 is a structural component of the dermal-epidermal junction defined its founding role in skin cohesion.","evidence":"Immunoelectron microscopy, radioimmunoprecipitation, and in situ antibody-induced blistering in human skin and primary keratinocytes","pmids":["8618013"],"confidence":"High","gaps":["Molecular partners at the anchoring filament not defined","Mechanism of secretion and assembly not addressed"]},{"year":1997,"claim":"Linking LAD1 loss to a blistering disease phenotype tested its functional importance in vivo in humans.","evidence":"Indirect immunofluorescence and radioimmunoprecipitation across a cohort of GABEB patient keratinocyte cultures","pmids":["9284104"],"confidence":"Medium","gaps":["Co-occurrence with BP180 abnormality leaves causality unresolved","No causative LAD1 mutation defined"]},{"year":1999,"claim":"Determining the oligomeric state showed LAD1 forms its own homotrimer rather than mixing with the related BP180.","evidence":"Two-dimensional SDS-PAGE under temperature-controlled dissociation with immunoblotting in human skin and keratinocytes","pmids":["9886264"],"confidence":"Medium","gaps":["Structural basis of trimerization not determined","Functional consequence of trimerization untested"]},{"year":2010,"claim":"Identifying LAD1 as a MEKK2 adaptor revealed an intracellular signaling function distinct from its structural role.","evidence":"Reciprocal co-immunoprecipitation, in vitro direct binding assay, calcium modulation, and nuclear translocation imaging","pmids":["20830310"],"confidence":"Medium","gaps":["Calcium-dependence mechanism not resolved at residue level","Connection to LAD1's structural pool unclear"]},{"year":2018,"claim":"Connecting LAD1 to EGF-induced filamin binding and actin dynamics established it as an effector linking EGF signaling to cytoskeletal regulation and proliferation/migration.","evidence":"SILAC phosphoproteomics, yeast two-hybrid, proximity ligation, co-IP, cosedimentation, shRNA knockdown, and xenograft tumor model in mammary cells","pmids":["29382783"],"confidence":"High","gaps":["Phosphosite functional roles not fully mapped","Relationship between filamin and MEKK2 binding modes unaddressed"]},{"year":2020,"claim":"Loss-of-function in colorectal cancer separated LAD1's role in metastasis from proliferation, defining a specific pro-migratory function.","evidence":"shRNA depletion with migration/invasion assays and an in vivo spleen-to-liver metastasis mouse model","pmids":["33267790"],"confidence":"Medium","gaps":["Molecular effectors in colorectal context not identified","No rescue with catalytically/structurally defined mutants"]},{"year":2023,"claim":"Identifying competitive Vimentin binding gave a molecular mechanism for how LAD1 stabilizes an EMT effector to promote cancer progression.","evidence":"MS interactome, co-IP, ubiquitination assays, and PDX/CDX xenograft gain/loss experiments in gastric cancer","pmids":["37718450"],"confidence":"Medium","gaps":["Binding interface with Vimentin/MAEA not mapped","Whether this mechanism operates in non-gastric contexts untested"]},{"year":2025,"claim":"Coupling LAD1 to cortactin/N-WASP phosphorylation and PI3K/AKT extended its cytoskeletal-remodeling role to esophageal carcinoma.","evidence":"Co-IP of a LAD1-LINC01305 interaction, phosphorylation and EMT marker assays, and migration/invasion functional readouts in ESCC","pmids":["39835575"],"confidence":"Low","gaps":["Limited mechanistic detail without full reconstitution","Direct vs indirect LAD1-lncRNA binding not validated reciprocally","Causal ordering of phosphorylation events unclear"]},{"year":null,"claim":"How LAD1's secreted structural anchoring function mechanistically relates to its intracellular signaling/adaptor roles remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structure of LAD1 or its complexes","No unifying account of how one protein performs extracellular anchoring and intracellular adaptor functions","Tissue-specific switch between roles undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,4]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,4]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[0]}],"complexes":["LAD1 homotrimer"],"partners":["MEKK2","FLNA","FLNB","SFN","VIM","MAEA","CTTN"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O00515","full_name":"Ladinin-1","aliases":["Linear IgA disease antigen","LADA"],"length_aa":517,"mass_kda":57.1,"function":"Anchoring filament protein which is a component of the basement membrane zone","subcellular_location":"Secreted, extracellular space, extracellular matrix, basement membrane","url":"https://www.uniprot.org/uniprotkb/O00515/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/LAD1","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/LAD1","total_profiled":1310},"omim":[{"mim_id":"612840","title":"LEUKOCYTE ADHESION DEFICIENCY, TYPE III; LAD3","url":"https://www.omim.org/entry/612840"},{"mim_id":"612268","title":"TUBULIN TYROSINE LIGASE-LIKE 5; TTLL5","url":"https://www.omim.org/entry/612268"},{"mim_id":"607901","title":"FERM DOMAIN-CONTAINING KINDLIN 3; FERMT3","url":"https://www.omim.org/entry/607901"},{"mim_id":"602314","title":"LADININ 1; LAD1","url":"https://www.omim.org/entry/602314"},{"mim_id":"600065","title":"INTEGRIN, BETA-2; ITGB2","url":"https://www.omim.org/entry/600065"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Actin filaments","reliability":"Enhanced"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"esophagus","ntpm":349.5},{"tissue":"skin 1","ntpm":207.4}],"url":"https://www.proteinatlas.org/search/LAD1"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"O00515","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O00515","model_url":"https://alphafold.ebi.ac.uk/files/AF-O00515-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O00515-F1-predicted_aligned_error_v6.png","plddt_mean":52.84},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=LAD1","jax_strain_url":"https://www.jax.org/strain/search?query=LAD1"},"sequence":{"accession":"O00515","fasta_url":"https://rest.uniprot.org/uniprotkb/O00515.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O00515/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O00515"}},"corpus_meta":[{"pmid":"8618013","id":"PMC_8618013","title":"LAD-1, the linear IgA bullous dermatosis autoantigen, is a novel 120-kDa anchoring filament protein synthesized by epidermal cells.","date":"1996","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/8618013","citation_count":186,"is_preprint":false},{"pmid":"11502758","id":"PMC_11502758","title":"LAD-1, the Caenorhabditis elegans L1CAM homologue, participates in embryonic and gonadal morphogenesis and is a substrate for fibroblast growth factor receptor pathway-dependent phosphotyrosine-based signaling.","date":"2001","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/11502758","citation_count":100,"is_preprint":false},{"pmid":"10936446","id":"PMC_10936446","title":"Genetic analysis of integrin function in man: LAD-1 and other syndromes.","date":"2000","source":"Matrix biology : journal of the International Society for Matrix Biology","url":"https://pubmed.ncbi.nlm.nih.gov/10936446","citation_count":81,"is_preprint":false},{"pmid":"25605789","id":"PMC_25605789","title":"Long-Term Balancing Selection in LAD1 Maintains a Missense Trans-Species Polymorphism in Humans, Chimpanzees, and Bonobos.","date":"2015","source":"Molecular biology and evolution","url":"https://pubmed.ncbi.nlm.nih.gov/25605789","citation_count":67,"is_preprint":false},{"pmid":"17875809","id":"PMC_17875809","title":"Reversion mutations in patients with leukocyte adhesion deficiency type-1 (LAD-1).","date":"2007","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/17875809","citation_count":61,"is_preprint":false},{"pmid":"14555469","id":"PMC_14555469","title":"D-xylose metabolism in Hypocrea jecorina: loss of the xylitol dehydrogenase step can be partially compensated for by lad1-encoded L-arabinitol-4-dehydrogenase.","date":"2003","source":"Eukaryotic cell","url":"https://pubmed.ncbi.nlm.nih.gov/14555469","citation_count":51,"is_preprint":false},{"pmid":"29382783","id":"PMC_29382783","title":"SILAC identifies LAD1 as a filamin-binding regulator of actin dynamics in response to EGF and a marker of aggressive breast tumors.","date":"2018","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/29382783","citation_count":48,"is_preprint":false},{"pmid":"27756787","id":"PMC_27756787","title":"A Four-Gene Promoter Methylation Marker Panel Consisting of GREM1, NEURL, LAD1, and NEFH Predicts Survival of Clear Cell Renal Cell Cancer Patients.","date":"2016","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/27756787","citation_count":45,"is_preprint":false},{"pmid":"21743503","id":"PMC_21743503","title":"Reduced-intensity conditioning hematopoietic SCT for pediatric patients with LAD-1: clinical efficacy and importance of chimerism.","date":"2011","source":"Bone marrow transplantation","url":"https://pubmed.ncbi.nlm.nih.gov/21743503","citation_count":28,"is_preprint":false},{"pmid":"29548898","id":"PMC_29548898","title":"Long term outcome of eight patients with type 1 Leukocyte Adhesion Deficiency (LAD-1): Not only infections, but high risk of autoimmune complications.","date":"2018","source":"Clinical immunology (Orlando, Fla.)","url":"https://pubmed.ncbi.nlm.nih.gov/29548898","citation_count":27,"is_preprint":false},{"pmid":"17078873","id":"PMC_17078873","title":"Dendritic cell-mediated HIV-1 transmission to T cells of LAD-1 patients is impaired due to the defect in LFA-1.","date":"2006","source":"Retrovirology","url":"https://pubmed.ncbi.nlm.nih.gov/17078873","citation_count":26,"is_preprint":false},{"pmid":"32080965","id":"PMC_32080965","title":"MSCs rescue impaired wound healing in a murine LAD1 model by adaptive responses to low TGF-β1 levels.","date":"2020","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/32080965","citation_count":23,"is_preprint":false},{"pmid":"9284104","id":"PMC_9284104","title":"LAD-1 is absent in a subset of junctional epidermolysis bullosa patients.","date":"1997","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/9284104","citation_count":23,"is_preprint":false},{"pmid":"12377933","id":"PMC_12377933","title":"Defective migration of monocyte-derived dendritic cells in LAD-1 immunodeficiency.","date":"2002","source":"Journal of leukocyte biology","url":"https://pubmed.ncbi.nlm.nih.gov/12377933","citation_count":21,"is_preprint":false},{"pmid":"32011724","id":"PMC_32011724","title":"Autoantibody Profile of a Cohort of 54 Italian Patients with Linear IgA Bullous Dermatosis: LAD-1 Denoted as a Major Auto-antigen of the Lamina Lucida Subtype.","date":"2020","source":"Acta dermato-venereologica","url":"https://pubmed.ncbi.nlm.nih.gov/32011724","citation_count":21,"is_preprint":false},{"pmid":"20830310","id":"PMC_20830310","title":"Calcium regulation of EGF-induced ERK5 activation: role of Lad1-MEKK2 interaction.","date":"2010","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/20830310","citation_count":19,"is_preprint":false},{"pmid":"33267790","id":"PMC_33267790","title":"LAD1 expression is associated with the metastatic potential of colorectal cancer cells.","date":"2020","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/33267790","citation_count":18,"is_preprint":false},{"pmid":"33536022","id":"PMC_33536022","title":"circ-ANXA7 facilitates lung adenocarcinoma progression via miR-331/LAD1 axis.","date":"2021","source":"Cancer cell international","url":"https://pubmed.ncbi.nlm.nih.gov/33536022","citation_count":18,"is_preprint":false},{"pmid":"37718450","id":"PMC_37718450","title":"LAD1 promotes malignant progression by diminishing ubiquitin-dependent degradation of vimentin in gastric cancer.","date":"2023","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37718450","citation_count":15,"is_preprint":false},{"pmid":"32279896","id":"PMC_32279896","title":"β2 Integrin Gene (ITGB2) mutation spectra in Pakistani families with leukocyte adhesion deficiency type 1 (LAD1).","date":"2020","source":"Immunobiology","url":"https://pubmed.ncbi.nlm.nih.gov/32279896","citation_count":15,"is_preprint":false},{"pmid":"25527966","id":"PMC_25527966","title":"Successful adjunctive immunoglobulin treatment in patients affected by leukocyte adhesion deficiency type 1 (LAD-1).","date":"2015","source":"Immunologic research","url":"https://pubmed.ncbi.nlm.nih.gov/25527966","citation_count":14,"is_preprint":false},{"pmid":"9886264","id":"PMC_9886264","title":"Type XVII collagen (BP180) and LAD-1 are present as separate trimeric complexes.","date":"1999","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/9886264","citation_count":13,"is_preprint":false},{"pmid":"25514840","id":"PMC_25514840","title":"Characterization of single amino acid substitutions in the β2 integrin subunit of patients with leukocyte adhesion deficiency (LAD)-1.","date":"2014","source":"Blood cells, molecules & diseases","url":"https://pubmed.ncbi.nlm.nih.gov/25514840","citation_count":12,"is_preprint":false},{"pmid":"34516317","id":"PMC_34516317","title":"High expression of ladinin-1 (LAD1) predicts adverse outcomes: a new candidate docetaxel resistance gene for prostatic cancer (PCa).","date":"2021","source":"Bioengineered","url":"https://pubmed.ncbi.nlm.nih.gov/34516317","citation_count":11,"is_preprint":false},{"pmid":"21195692","id":"PMC_21195692","title":"A novel 3' splice-site mutation and a novel gross deletion in leukocyte adhesion deficiency (LAD)-1.","date":"2010","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/21195692","citation_count":11,"is_preprint":false},{"pmid":"25135596","id":"PMC_25135596","title":"Successful anti-TNF-α treatment in a girl with LAD-1 disease and autoimmune manifestations.","date":"2014","source":"Journal of clinical 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carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/39835575","citation_count":1,"is_preprint":false},{"pmid":"36633773","id":"PMC_36633773","title":"Circ_0060937 Contributes to the Development of Lung Cancer via Positively Regulating LAD1 Expression by Binding to miR-1304-5p.","date":"2023","source":"Biochemical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/36633773","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16185,"output_tokens":2091,"usd":0.03996,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9078,"output_tokens":2720,"usd":0.056695,"stage2_stop_reason":"end_turn"},"total_usd":0.096655,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"LAD-1 (Ladinin-1) is a novel 120-kDa anchoring filament protein synthesized and secreted by epidermal keratinocytes, localizing exclusively to anchoring filaments at the dermal-epidermal junction as determined by immunoelectron microscopy. A monoclonal antibody against LAD-1 induced dermal-epidermal separation of human skin in situ, establishing its role in maintaining dermal-epidermal cohesion.\",\n      \"method\": \"Radioimmunoprecipitation, immunoelectron microscopy, immunoblot, in situ antibody-induced blistering assay, primary keratinocyte culture\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (immunoelectron microscopy, radioimmunoprecipitation, functional in situ blistering assay) in a single rigorous characterization study\",\n      \"pmids\": [\"8618013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"LAD-1 exists as a high-molecular-mass homotrimeric complex (analogous to BP180 trimers) in human skin and keratinocytes, and does not form mixed complexes with BP180, as shown by two-dimensional electrophoresis under temperature-dissociation conditions.\",\n      \"method\": \"Two-dimensional SDS-PAGE with temperature-controlled dissociation, immunoblotting, silver staining\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal methods (2D gel + immunoblot) in a single lab study definitively showing homotrimeric complex and absence of BP180 mixed complexes\",\n      \"pmids\": [\"9886264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"LAD-1 protein expression is absent or greatly reduced in the majority of generalized atrophic benign epidermolysis bullosa (GABEB) patients, and this defect co-occurs with abnormal BP180 expression, as confirmed by indirect immunofluorescence and radioimmunoprecipitation from cultured keratinocytes.\",\n      \"method\": \"Indirect immunofluorescence microscopy, radioimmunoprecipitation from keratinocyte cultures\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal methods (immunofluorescence + radioimmunoprecipitation) in a single lab with a cohort of 44 patients\",\n      \"pmids\": [\"9284104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"LAD1 (Ladinin-1) functions as an adaptor protein in the EGF-to-ERK5 signaling pathway: EGF stimulation increases MEKK2 binding to LAD1 in a calcium-dependent manner, and this direct interaction (demonstrated by in vitro binding assay) is required for MEKK2 nuclear translocation and full tyrosine phosphorylation of MEKK2, thereby enabling ERK5 activation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assay, intracellular calcium modifiers, nuclear translocation imaging\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP plus in vitro direct binding assay in a single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"20830310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"LAD1 (Ladinin-1) is transcriptionally induced by EGF, phosphorylated downstream of EGF signaling, and binds to actin-crosslinking filamin proteins; it co-localizes with actin stress fibers and participates in actin dynamics in collaboration with 14-3-3σ (SFN), mediating mammary cell proliferation and migration.\",\n      \"method\": \"Phosphoproteomics (SILAC), yeast two-hybrid, proximity ligation assay, co-immunoprecipitation, cosedimentation assay, shRNA knockdown, xenograft tumor model\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (SILAC phosphoproteomics, yeast two-hybrid, PLA, co-IP, cosedimentation, in vivo xenograft) in a single rigorous study\",\n      \"pmids\": [\"29382783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LAD1 promotes gastric cancer progression by competitively binding to Vimentin, preventing Vimentin's interaction with the E3 ubiquitin ligase MAEA, thereby reducing K48-linked ubiquitination and degradation of Vimentin, leading to increased Vimentin protein levels.\",\n      \"method\": \"Mass spectrometry (interactome), co-immunoprecipitation, ubiquitination assay, in vitro and in vivo functional gain/loss experiments (PDX and CDX xenograft models)\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, MS-based interactome identification, and ubiquitination assay in a single lab with in vivo validation\",\n      \"pmids\": [\"37718450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"LAD1 (Ladinin-1) co-regulates cortactin (CTTN) and N-WASP phosphorylation to mediate cytoskeletal reorganization in esophageal squamous cell carcinoma (ESCC), and together with LINC01305 promotes EMT via activation of the PI3K/AKT signaling pathway.\",\n      \"method\": \"Co-IP (LAD1-LINC01305 binding), phosphorylation assays for CTTN and N-WASP, EMT marker analysis, PI3K/AKT pathway assays, functional migration/invasion assays\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, mechanistic details limited in abstract; binding to a lncRNA and downstream phosphorylation readouts without full reconstitution\",\n      \"pmids\": [\"39835575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"LAD1 depletion inhibits migration and invasion of colorectal cancer cells (SW620, Caco-2) and reduces liver metastasis in a spleen-to-liver mouse model, without affecting cell proliferation, indicating a specific role in metastatic behavior.\",\n      \"method\": \"shRNA-mediated LAD1 depletion, migration/invasion assays, in vivo spleen-to-liver metastasis mouse model, immunohistochemistry\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined loss-of-function phenotype with both in vitro and in vivo readouts in a single lab\",\n      \"pmids\": [\"33267790\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"LAD1 (Ladinin-1) is a homotrimeric anchoring filament protein secreted by keratinocytes that maintains dermal-epidermal cohesion; it also acts as an intracellular signaling adaptor that binds MEKK2 in a calcium-dependent manner to relay EGF signals to ERK5, binds filamin proteins to regulate actin dynamics and cell migration downstream of EGF, and in cancer contexts protects Vimentin from MAEA-mediated ubiquitin-dependent degradation, collectively promoting cell migration, invasion, and metastasis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"LAD1 (Ladinin-1) is a 120-kDa anchoring filament protein originally characterized as a keratinocyte-secreted component of the dermal-epidermal junction, where antibody-induced disruption causes dermal-epidermal separation, establishing its role in maintaining dermal-epidermal cohesion [#0]. In skin it assembles as a homotrimeric complex distinct from BP180 [#1], and its loss accompanies abnormal BP180 expression in generalized atrophic benign epidermolysis bullosa patients [#2]. Beyond its structural role, LAD1 acts as an intracellular signaling adaptor downstream of EGF: it binds MEKK2 in a calcium-dependent manner to enable MEKK2 nuclear translocation, tyrosine phosphorylation, and ERK5 activation [#3], and is itself transcriptionally induced and phosphorylated by EGF signaling, binding actin-crosslinking filamin proteins and co-localizing with actin stress fibers together with 14-3-3σ to drive cell proliferation and migration [#4]. In cancer, LAD1 promotes migratory and metastatic behavior — its depletion blocks colorectal cancer migration, invasion, and liver metastasis without affecting proliferation [#7] — and it stabilizes Vimentin by competitively blocking the MAEA E3 ligase, reducing K48-linked ubiquitination and degradation of Vimentin in gastric cancer [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Establishing whether LAD1 is a structural component of the dermal-epidermal junction defined its founding role in skin cohesion.\",\n      \"evidence\": \"Immunoelectron microscopy, radioimmunoprecipitation, and in situ antibody-induced blistering in human skin and primary keratinocytes\",\n      \"pmids\": [\"8618013\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular partners at the anchoring filament not defined\", \"Mechanism of secretion and assembly not addressed\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Linking LAD1 loss to a blistering disease phenotype tested its functional importance in vivo in humans.\",\n      \"evidence\": \"Indirect immunofluorescence and radioimmunoprecipitation across a cohort of GABEB patient keratinocyte cultures\",\n      \"pmids\": [\"9284104\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Co-occurrence with BP180 abnormality leaves causality unresolved\", \"No causative LAD1 mutation defined\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Determining the oligomeric state showed LAD1 forms its own homotrimer rather than mixing with the related BP180.\",\n      \"evidence\": \"Two-dimensional SDS-PAGE under temperature-controlled dissociation with immunoblotting in human skin and keratinocytes\",\n      \"pmids\": [\"9886264\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of trimerization not determined\", \"Functional consequence of trimerization untested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identifying LAD1 as a MEKK2 adaptor revealed an intracellular signaling function distinct from its structural role.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, in vitro direct binding assay, calcium modulation, and nuclear translocation imaging\",\n      \"pmids\": [\"20830310\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Calcium-dependence mechanism not resolved at residue level\", \"Connection to LAD1's structural pool unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Connecting LAD1 to EGF-induced filamin binding and actin dynamics established it as an effector linking EGF signaling to cytoskeletal regulation and proliferation/migration.\",\n      \"evidence\": \"SILAC phosphoproteomics, yeast two-hybrid, proximity ligation, co-IP, cosedimentation, shRNA knockdown, and xenograft tumor model in mammary cells\",\n      \"pmids\": [\"29382783\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphosite functional roles not fully mapped\", \"Relationship between filamin and MEKK2 binding modes unaddressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Loss-of-function in colorectal cancer separated LAD1's role in metastasis from proliferation, defining a specific pro-migratory function.\",\n      \"evidence\": \"shRNA depletion with migration/invasion assays and an in vivo spleen-to-liver metastasis mouse model\",\n      \"pmids\": [\"33267790\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular effectors in colorectal context not identified\", \"No rescue with catalytically/structurally defined mutants\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identifying competitive Vimentin binding gave a molecular mechanism for how LAD1 stabilizes an EMT effector to promote cancer progression.\",\n      \"evidence\": \"MS interactome, co-IP, ubiquitination assays, and PDX/CDX xenograft gain/loss experiments in gastric cancer\",\n      \"pmids\": [\"37718450\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding interface with Vimentin/MAEA not mapped\", \"Whether this mechanism operates in non-gastric contexts untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Coupling LAD1 to cortactin/N-WASP phosphorylation and PI3K/AKT extended its cytoskeletal-remodeling role to esophageal carcinoma.\",\n      \"evidence\": \"Co-IP of a LAD1-LINC01305 interaction, phosphorylation and EMT marker assays, and migration/invasion functional readouts in ESCC\",\n      \"pmids\": [\"39835575\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Limited mechanistic detail without full reconstitution\", \"Direct vs indirect LAD1-lncRNA binding not validated reciprocally\", \"Causal ordering of phosphorylation events unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How LAD1's secreted structural anchoring function mechanistically relates to its intracellular signaling/adaptor roles remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of LAD1 or its complexes\", \"No unifying account of how one protein performs extracellular anchoring and intracellular adaptor functions\", \"Tissue-specific switch between roles undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [\"LAD1 homotrimer\"],\n    \"partners\": [\"MEKK2\", \"FLNA\", \"FLNB\", \"SFN\", \"VIM\", \"MAEA\", \"CTTN\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}