{"gene":"GPC4","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":1995,"finding":"GPC4 (K-glypican) is a GPI-anchored heparan sulfate proteoglycan (HSPG); transfection of epitope-tagged full-length GPC4 cDNA into MDCK cells confirmed it is expressed as a GPI-anchored HSPG at the cell surface, with heparan sulfate attachment sites and a GPI anchor in its C-terminal region.","method":"cDNA transfection in MDCK cells, molecular characterization of GPI anchor and HS attachment sites","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — direct biochemical demonstration in transfected cells, foundational paper with 129 citations","pmids":["7657705"],"is_preprint":false},{"year":2000,"finding":"A GPC3 missense mutation (W296R) that also affects a conserved residue in all glypicans including GPC4 causes poor processing and failure to increase cell surface heparan sulfate expression, illustrating the functional importance of this conserved residue for glypican surface presentation.","method":"Recombinant mutant protein expression and functional assay of cell surface heparan sulfate","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 — functional assay of recombinant protein, but finding is primarily about GPC3 with GPC4 relevance inferred from conservation","pmids":["10814714"],"is_preprint":false},{"year":2019,"finding":"CD36 interacts with GPC4 and promotes proteasome-dependent ubiquitination and degradation of GPC4, thereby inhibiting β-catenin/c-myc signaling and suppressing downstream glycolytic target genes GLUT1, HK2, PKM2, and LDHA in colorectal cancer cells.","method":"Co-immunoprecipitation (CD36-GPC4 interaction), proteasome inhibitor assays, loss-of-function knockdown, in vitro and in vivo tumor models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, proteasome inhibitor rescue, in vitro and in vivo functional validation; 198 citations","pmids":["31484922"],"is_preprint":false},{"year":2019,"finding":"GPC4 loss-of-function variants (truncating mutations eliminating N-linked glycosylation at Asn514 and the GPI anchor site at Ser529) cause Keipert syndrome; recombinant truncated proteins p.Gln506* and p.Glu496* were less stable than wild type, and Gpc4 knockout mice displayed craniofacial and digital abnormalities consistent with the human syndrome.","method":"Whole-exome sequencing, recombinant protein stability assays, Gpc4 knockout mouse phenotyping","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 1-2 — functional studies of recombinant protein combined with KO mouse phenotype and human genetics","pmids":["30982611"],"is_preprint":false},{"year":2016,"finding":"In zebrafish, gpc4 acts in the non-canonical Wnt signaling pathway in chondrocytes to regulate convergent-extension during palate morphogenesis; gpc4 mutants show defective cell intercalation, and genetic dissection indicates gpc4 functions in receiving chondrocytes juxtaposed to Wnt-secreting ectoderm.","method":"Zebrafish gpc4 mutant analysis, genetic epistasis with wnt5b, wnt9a, wls and frzb mutants","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with multiple pathway components, replicated across two studies","pmids":["27287801"],"is_preprint":false},{"year":2016,"finding":"In zebrafish, gpc4 is required for non-canonical Wnt signaling to regulate the timing of chondrocyte maturation and onset of endochondral ossification; loss of gpc4 causes severely delayed endochondral ossification in Meckel's cartilage.","method":"Zebrafish gpc4 mutant analysis, comparison with wls, wnt5b, and wnt9a mutants","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis, multiple pathway mutants, consistent with companion study","pmids":["27908786"],"is_preprint":false},{"year":2022,"finding":"FTO-mediated m6A demethylation regulates GPC4 mRNA stability via the m6A reader YTHDF3; reduced FTO increases m6A modification of GPC4 mRNA, decreasing GPC4 expression, while GPC4 in turn activates TLR4/NF-κB inflammatory signaling in microglia during autoimmune uveitis.","method":"RNA stability assays, RNA-seq, siRNA knockdown, FTO inhibitor treatment, rescue experiments","journal":"Genes & diseases","confidence":"Medium","confidence_rationale":"Tier 2-3 — RNA stability assay and rescue experiments, single lab but multiple methods","pmids":["37492748"],"is_preprint":false},{"year":2024,"finding":"GPC4 promotes HS3ST1-mediated glycolysis in lung adenocarcinoma; the interaction between HS3ST1 and GPC4 was demonstrated by immunoprecipitation.","method":"Immunoprecipitation of HS3ST1-GPC4 interaction, glycolysis assays in LUAD cell lines","journal":"Cancers","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP, limited mechanistic follow-up","pmids":["38398086"],"is_preprint":false},{"year":2025,"finding":"Aβ fibrils induce upregulation of GPC4 on the microglia surface; shed GPC4 facilitates tau aggregate uptake and seeding in neurons in trans, and these effects are amplified by APOE; GPC4 enhances microglia phagocytosis of tau aggregates in cell culture; glial GPC4 expression exacerbates motor deficits and reduces lifespan in a Drosophila amyloidosis model.","method":"Microglia surfaceome profiling, cell culture tau uptake/seeding assays, Drosophila in vivo model, APOE co-treatment","journal":"Molecular neurodegeneration","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (surfaceomics, in vitro seeding, Drosophila in vivo), single lab","pmids":["40883746"],"is_preprint":false},{"year":2025,"finding":"GPC4 knockdown suppresses TLR4/NF-κB signaling and reverses pro-inflammatory effects in astrocytes in ischemic stroke models; GPC4 and TLR4 are co-expressed in astrocytes and function together in the same inflammatory axis.","method":"siRNA knockdown of GPC4, Western blot, RT-qPCR, immunofluorescence co-localization, molecular docking","journal":"Journal of ethnopharmacology","confidence":"Medium","confidence_rationale":"Tier 2-3 — siRNA KD with defined signaling phenotype, multiple validation methods, single lab","pmids":["41724289"],"is_preprint":false},{"year":2025,"finding":"CCN1 (a secreted matricellular protein from radial glial cells) directly interacts with GPC4 on radial glia, and this interaction is required for GPC4 to maintain neural stem cells through the Sonic Hedgehog (Shh) signaling pathway in a heparin-binding-dependent manner.","method":"Co-immunoprecipitation/interaction assays, Ccn1 loss-of-function in radial glia, Shh pathway readout, heparin binding assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2-3 — interaction and loss-of-function with pathway readout, preprint only","pmids":["bio_10.1101_2025.05.16.654402"],"is_preprint":true},{"year":2024,"finding":"GPC4 co-localizes with Vangl2 presynaptically at mossy fiber boutons in the hippocampus and mediates stabilization of the postsynaptic orphan receptor GPR158; Vangl2-dependent planar cell polarity signaling requires GPC4 to maintain mossy fiber bouton/thorny excrescence synapse morphology and function.","method":"Co-localization by immunofluorescence, Vangl2 knockout mouse synapse morphology and electrophysiology","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 — co-localization without direct GPC4 manipulation, preprint","pmids":["bio_10.1101_2024.05.28.596141"],"is_preprint":true}],"current_model":"GPC4 is a GPI-anchored heparan sulfate proteoglycan that modulates cell signaling at the cell surface: it acts as a co-receptor in non-canonical Wnt/planar cell polarity signaling (regulating convergent-extension and chondrocyte maturation), is ubiquitinated and degraded by CD36 to suppress β-catenin/c-myc-driven glycolysis, interacts with CCN1 to sustain Shh-dependent neural stem cell maintenance, mediates TLR4/NF-κB inflammatory signaling in glia, and—when shed or upregulated by Aβ in microglia—facilitates tau aggregate seeding and neurodegeneration."},"narrative":{"teleology":[{"year":1995,"claim":"Establishing GPC4's molecular identity as a GPI-anchored heparan sulfate proteoglycan resolved what class of molecule GPC4 encodes and how it is tethered to the cell surface.","evidence":"cDNA transfection in MDCK cells with biochemical characterization of GPI anchor and HS attachment sites","pmids":["7657705"],"confidence":"High","gaps":["No signaling function yet assigned","No in vivo requirement demonstrated","Heparan sulfate chain composition and length uncharacterized"]},{"year":2000,"claim":"Demonstration that a conserved tryptophan residue is required for glypican processing and surface HS presentation revealed a structural requirement shared across the glypican family including GPC4.","evidence":"Recombinant GPC3 W296R mutant expression and surface HS functional assay, with conservation-based inference to GPC4","pmids":["10814714"],"confidence":"Medium","gaps":["Direct mutagenesis of GPC4 at this residue not performed","Processing mechanism (protease identity, ER/Golgi steps) undefined"]},{"year":2016,"claim":"Genetic epistasis in zebrafish revealed that GPC4 functions specifically in non-canonical Wnt signaling to drive convergent-extension cell intercalation in chondrocytes and to regulate the timing of endochondral ossification, establishing GPC4 as a co-receptor in PCP signaling.","evidence":"Zebrafish gpc4 mutant analysis with epistasis to wnt5b, wnt9a, wls, and frzb mutants across two independent studies","pmids":["27287801","27908786"],"confidence":"High","gaps":["Direct biochemical interaction between GPC4 and Wnt ligands not shown","Whether heparan sulfate chains are essential for PCP signal transduction not tested","Mammalian in vivo PCP role not directly demonstrated"]},{"year":2019,"claim":"Identification of CD36 as a GPC4 interactor that triggers its proteasomal degradation revealed a mechanism by which GPC4 protein levels are controlled and linked GPC4 abundance to β-catenin/c-myc-driven glycolytic reprogramming in colorectal cancer.","evidence":"Reciprocal Co-IP, proteasome inhibitor rescue, knockdown, and in vivo tumor xenograft models","pmids":["31484922"],"confidence":"High","gaps":["E3 ubiquitin ligase responsible for GPC4 ubiquitination not identified","Whether GPC4 directly binds β-catenin or acts via an intermediate not resolved","How CD36-GPC4 interaction triggers ubiquitination mechanistically unclear"]},{"year":2019,"claim":"Human genetics linked GPC4 loss-of-function truncating mutations to Keipert syndrome and Gpc4 knockout mice recapitulated the craniofacial-digital phenotype, establishing GPC4 as the causal gene for this Mendelian disorder.","evidence":"Whole-exome sequencing of affected families, recombinant protein stability assays, Gpc4 KO mouse phenotyping","pmids":["30982611"],"confidence":"High","gaps":["Which signaling pathway (Wnt, Shh, FGF) is primarily disrupted in Keipert syndrome not determined","Whether partial loss-of-function alleles produce milder phenotypes unknown"]},{"year":2022,"claim":"Showing that GPC4 activates TLR4/NF-κB inflammatory signaling in microglia, with its own mRNA stability regulated by FTO-mediated m6A demethylation via YTHDF3, connected GPC4 to innate immune activation and revealed an epitranscriptomic layer of GPC4 regulation.","evidence":"RNA stability assays, siRNA knockdown, FTO inhibitor treatment, and rescue experiments in microglia during autoimmune uveitis","pmids":["37492748"],"confidence":"Medium","gaps":["Whether GPC4 directly binds TLR4 or acts through HS-mediated ligand presentation not distinguished","Single-lab finding without independent replication","In vivo validation of m6A-GPC4 axis limited"]},{"year":2025,"claim":"Multiple studies converged to show GPC4 functions in neuroinflammation and neurodegeneration: Aβ-induced GPC4 upregulation and shedding from microglia facilitates tau seeding in neurons, and GPC4 knockdown in astrocytes suppresses TLR4/NF-κB signaling in ischemic stroke.","evidence":"Microglia surfaceome profiling, tau uptake/seeding assays, Drosophila in vivo amyloidosis model, astrocyte siRNA knockdown with signaling readouts","pmids":["40883746","41724289"],"confidence":"Medium","gaps":["Molecular mechanism of GPC4 shedding (sheddase identity) not identified","Whether GPC4-mediated tau seeding is HS-dependent not tested","In vivo mammalian validation of GPC4 in tauopathy lacking"]},{"year":null,"claim":"Key unresolved questions include the structural basis of GPC4 co-receptor function, the identity of the E3 ligase mediating its ubiquitination, whether HS chains are required for each of its signaling roles, and how GPC4 shedding is regulated in neurodegeneration.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal or cryo-EM structure of GPC4 or GPC4–ligand complexes","HS chain requirements not dissected for individual signaling pathways","Sheddase(s) responsible for GPC4 release not identified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,4,5,6,9]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,3,8]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,4,5,6,9]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[6,9]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[3,4,5]}],"complexes":[],"partners":["CD36","TLR4","VANGL2","HS3ST1","CCN1"],"other_free_text":[]},"mechanistic_narrative":"GPC4 is a GPI-anchored heparan sulfate proteoglycan that functions as a cell-surface co-receptor modulating multiple signaling pathways, including non-canonical Wnt/planar cell polarity, β-catenin, TLR4/NF-κB, and Sonic Hedgehog signaling. In zebrafish, GPC4 is required for non-canonical Wnt signaling during convergent-extension cell movements and chondrocyte maturation in palate morphogenesis and endochondral ossification [PMID:27287801, PMID:27908786]. CD36 promotes proteasome-dependent ubiquitination and degradation of GPC4, thereby suppressing β-catenin/c-myc-driven glycolysis in colorectal cancer [PMID:31484922], while in glial cells GPC4 activates TLR4/NF-κB inflammatory signaling [PMID:37492748, PMID:41724289], and Aβ-induced shedding of GPC4 from microglia facilitates tau aggregate seeding in neurons [PMID:40883746]. Loss-of-function mutations in GPC4 that eliminate the GPI anchor and key glycosylation sites cause Keipert syndrome, a craniofacial-digital disorder recapitulated by Gpc4 knockout mice [PMID:30982611]."},"prefetch_data":{"uniprot":{"accession":"O75487","full_name":"Glypican-4","aliases":["K-glypican"],"length_aa":556,"mass_kda":62.4,"function":"Cell surface proteoglycan that bears heparan sulfate. May be involved in the development of kidney tubules and of the central nervous system (By similarity)","subcellular_location":"Secreted, extracellular space","url":"https://www.uniprot.org/uniprotkb/O75487/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/GPC4","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":[{"gene":"CANX","stoichiometry":0.2},{"gene":"SRP14","stoichiometry":0.2},{"gene":"SRP9","stoichiometry":0.2},{"gene":"TMED10","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/GPC4","total_profiled":1310},"omim":[{"mim_id":"604404","title":"GLYPICAN 6; GPC6","url":"https://www.omim.org/entry/604404"},{"mim_id":"600395","title":"GLYPICAN 1; GPC1","url":"https://www.omim.org/entry/600395"},{"mim_id":"312870","title":"SIMPSON-GOLABI-BEHMEL SYNDROME, TYPE 1; SGBS1","url":"https://www.omim.org/entry/312870"},{"mim_id":"301026","title":"KEIPERT SYNDROME; KPTS","url":"https://www.omim.org/entry/301026"},{"mim_id":"300168","title":"GLYPICAN 4; GPC4","url":"https://www.omim.org/entry/300168"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/GPC4"},"hgnc":{"alias_symbol":["K-glypican"],"prev_symbol":[]},"alphafold":{"accession":"O75487","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75487","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75487-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75487-F1-predicted_aligned_error_v6.png","plddt_mean":83.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GPC4","jax_strain_url":"https://www.jax.org/strain/search?query=GPC4"},"sequence":{"accession":"O75487","fasta_url":"https://rest.uniprot.org/uniprotkb/O75487.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75487/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75487"}},"corpus_meta":[{"pmid":"31484922","id":"PMC_31484922","title":"CD36 inhibits β-catenin/c-myc-mediated glycolysis through ubiquitination of GPC4 to repress colorectal tumorigenesis.","date":"2019","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/31484922","citation_count":198,"is_preprint":false},{"pmid":"7657705","id":"PMC_7657705","title":"K-glypican: a novel GPI-anchored heparan sulfate proteoglycan that is highly expressed in developing brain and kidney.","date":"1995","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/7657705","citation_count":129,"is_preprint":false},{"pmid":"10814714","id":"PMC_10814714","title":"Mutational analysis of the GPC3/GPC4 glypican gene cluster on Xq26 in patients with Simpson-Golabi-Behmel syndrome: identification of loss-of-function mutations in the GPC3 gene.","date":"2000","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/10814714","citation_count":95,"is_preprint":false},{"pmid":"9787072","id":"PMC_9787072","title":"GPC4, the gene for human K-glypican, flanks GPC3 on xq26: deletion of the GPC3-GPC4 gene cluster in one family with Simpson-Golabi-Behmel syndrome.","date":"1998","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/9787072","citation_count":71,"is_preprint":false},{"pmid":"10329016","id":"PMC_10329016","title":"GPC6, a novel member of the glypican gene family, encodes a product structurally related to GPC4 and is colocalized with GPC5 on human chromosome 13.","date":"1999","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/10329016","citation_count":60,"is_preprint":false},{"pmid":"27287801","id":"PMC_27287801","title":"Roles of Wnt pathway genes wls, wnt9a, wnt5b, frzb and gpc4 in regulating convergent-extension during zebrafish palate morphogenesis.","date":"2016","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/27287801","citation_count":41,"is_preprint":false},{"pmid":"27908786","id":"PMC_27908786","title":"Distinct requirements of wls, wnt9a, wnt5b and gpc4 in regulating chondrocyte maturation and timing of endochondral ossification.","date":"2016","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/27908786","citation_count":41,"is_preprint":false},{"pmid":"37492748","id":"PMC_37492748","title":"FTO-mediated m6A modification alleviates autoimmune uveitis by regulating microglia phenotypes via the GPC4/TLR4/NF-κB signaling axis.","date":"2022","source":"Genes & diseases","url":"https://pubmed.ncbi.nlm.nih.gov/37492748","citation_count":32,"is_preprint":false},{"pmid":"30982611","id":"PMC_30982611","title":"Pathogenic Variants in GPC4 Cause Keipert Syndrome.","date":"2019","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30982611","citation_count":24,"is_preprint":false},{"pmid":"30048822","id":"PMC_30048822","title":"Duplications of GPC3 and GPC4 genes in symptomatic female carriers of Simpson-Golabi-Behmel syndrome type 1.","date":"2018","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30048822","citation_count":12,"is_preprint":false},{"pmid":"38398086","id":"PMC_38398086","title":"Hypoxia-Derived Exosomes Promote Lung Adenocarcinoma by Regulating HS3ST1-GPC4-Mediated Glycolysis.","date":"2024","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/38398086","citation_count":10,"is_preprint":false},{"pmid":"40883746","id":"PMC_40883746","title":"β-Amyloid induces microglial expression of GPC4 and APOE leading to increased neuronal tau pathology and toxicity.","date":"2025","source":"Molecular neurodegeneration","url":"https://pubmed.ncbi.nlm.nih.gov/40883746","citation_count":2,"is_preprint":false},{"pmid":"31583675","id":"PMC_31583675","title":"Xq26 duplications lead to undergrowth or overgrowth via competing pathways including GPC3/GPC4.","date":"2019","source":"Annals of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31583675","citation_count":2,"is_preprint":false},{"pmid":"40060520","id":"PMC_40060520","title":"β-Amyloid Induces Microglial Expression of GPC4 and APOE Leading to Increased Neuronal Tau Pathology and Toxicity.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40060520","citation_count":1,"is_preprint":false},{"pmid":"41724289","id":"PMC_41724289","title":"From multi-omics discovery to experimental validation: GPC4 mediates the anti-neuroinflammatory effects of Buyang Huanwu decoction via TLR4/NF-κB signaling in ischemic stroke with Qi deficiency and blood stasis syndrome.","date":"2026","source":"Journal of ethnopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/41724289","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.05.16.654402","title":"CCN1 forms a complex with GPC4 and heparin to fine-tune signaling activities for cortical neural stem cell maintenance","date":"2025-05-20","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.16.654402","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.07.30.25332433","title":"Large-scale plasma proteomics uncovers preclinical molecular signatures of Parkinson’s disease and overlap with other neurodegenerative disorders","date":"2025-07-30","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.30.25332433","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.05.10.653258","title":"Functional targeting of Glypican-4 by a conformation-specific single-domain antibody","date":"2025-05-15","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.10.653258","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.05.28.596141","title":"The correct temporal connectivity of the DG-CA3 circuits involved in declarative memory processes depends on Vangl2-dependent planar cell polarity signaling","date":"2024-06-01","source":"bioRxiv","url":"https://doi.org/10.1101/2024.05.28.596141","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11320,"output_tokens":2776,"usd":0.0378},"stage2":{"model":"claude-opus-4-6","input_tokens":6147,"output_tokens":2205,"usd":0.12879},"total_usd":0.16659,"stage1_batch_id":"msgbatch_01DzrsgVgYngY1hvM3DU1yF2","stage2_batch_id":"msgbatch_01KA9b7iRzKFdhoBUT7vCvSe","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"GPC4 (K-glypican) is a GPI-anchored heparan sulfate proteoglycan (HSPG); transfection of epitope-tagged full-length GPC4 cDNA into MDCK cells confirmed it is expressed as a GPI-anchored HSPG at the cell surface, with heparan sulfate attachment sites and a GPI anchor in its C-terminal region.\",\n      \"method\": \"cDNA transfection in MDCK cells, molecular characterization of GPI anchor and HS attachment sites\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct biochemical demonstration in transfected cells, foundational paper with 129 citations\",\n      \"pmids\": [\"7657705\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"A GPC3 missense mutation (W296R) that also affects a conserved residue in all glypicans including GPC4 causes poor processing and failure to increase cell surface heparan sulfate expression, illustrating the functional importance of this conserved residue for glypican surface presentation.\",\n      \"method\": \"Recombinant mutant protein expression and functional assay of cell surface heparan sulfate\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional assay of recombinant protein, but finding is primarily about GPC3 with GPC4 relevance inferred from conservation\",\n      \"pmids\": [\"10814714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CD36 interacts with GPC4 and promotes proteasome-dependent ubiquitination and degradation of GPC4, thereby inhibiting β-catenin/c-myc signaling and suppressing downstream glycolytic target genes GLUT1, HK2, PKM2, and LDHA in colorectal cancer cells.\",\n      \"method\": \"Co-immunoprecipitation (CD36-GPC4 interaction), proteasome inhibitor assays, loss-of-function knockdown, in vitro and in vivo tumor models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, proteasome inhibitor rescue, in vitro and in vivo functional validation; 198 citations\",\n      \"pmids\": [\"31484922\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GPC4 loss-of-function variants (truncating mutations eliminating N-linked glycosylation at Asn514 and the GPI anchor site at Ser529) cause Keipert syndrome; recombinant truncated proteins p.Gln506* and p.Glu496* were less stable than wild type, and Gpc4 knockout mice displayed craniofacial and digital abnormalities consistent with the human syndrome.\",\n      \"method\": \"Whole-exome sequencing, recombinant protein stability assays, Gpc4 knockout mouse phenotyping\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — functional studies of recombinant protein combined with KO mouse phenotype and human genetics\",\n      \"pmids\": [\"30982611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In zebrafish, gpc4 acts in the non-canonical Wnt signaling pathway in chondrocytes to regulate convergent-extension during palate morphogenesis; gpc4 mutants show defective cell intercalation, and genetic dissection indicates gpc4 functions in receiving chondrocytes juxtaposed to Wnt-secreting ectoderm.\",\n      \"method\": \"Zebrafish gpc4 mutant analysis, genetic epistasis with wnt5b, wnt9a, wls and frzb mutants\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with multiple pathway components, replicated across two studies\",\n      \"pmids\": [\"27287801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In zebrafish, gpc4 is required for non-canonical Wnt signaling to regulate the timing of chondrocyte maturation and onset of endochondral ossification; loss of gpc4 causes severely delayed endochondral ossification in Meckel's cartilage.\",\n      \"method\": \"Zebrafish gpc4 mutant analysis, comparison with wls, wnt5b, and wnt9a mutants\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis, multiple pathway mutants, consistent with companion study\",\n      \"pmids\": [\"27908786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FTO-mediated m6A demethylation regulates GPC4 mRNA stability via the m6A reader YTHDF3; reduced FTO increases m6A modification of GPC4 mRNA, decreasing GPC4 expression, while GPC4 in turn activates TLR4/NF-κB inflammatory signaling in microglia during autoimmune uveitis.\",\n      \"method\": \"RNA stability assays, RNA-seq, siRNA knockdown, FTO inhibitor treatment, rescue experiments\",\n      \"journal\": \"Genes & diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — RNA stability assay and rescue experiments, single lab but multiple methods\",\n      \"pmids\": [\"37492748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GPC4 promotes HS3ST1-mediated glycolysis in lung adenocarcinoma; the interaction between HS3ST1 and GPC4 was demonstrated by immunoprecipitation.\",\n      \"method\": \"Immunoprecipitation of HS3ST1-GPC4 interaction, glycolysis assays in LUAD cell lines\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP, limited mechanistic follow-up\",\n      \"pmids\": [\"38398086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Aβ fibrils induce upregulation of GPC4 on the microglia surface; shed GPC4 facilitates tau aggregate uptake and seeding in neurons in trans, and these effects are amplified by APOE; GPC4 enhances microglia phagocytosis of tau aggregates in cell culture; glial GPC4 expression exacerbates motor deficits and reduces lifespan in a Drosophila amyloidosis model.\",\n      \"method\": \"Microglia surfaceome profiling, cell culture tau uptake/seeding assays, Drosophila in vivo model, APOE co-treatment\",\n      \"journal\": \"Molecular neurodegeneration\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (surfaceomics, in vitro seeding, Drosophila in vivo), single lab\",\n      \"pmids\": [\"40883746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GPC4 knockdown suppresses TLR4/NF-κB signaling and reverses pro-inflammatory effects in astrocytes in ischemic stroke models; GPC4 and TLR4 are co-expressed in astrocytes and function together in the same inflammatory axis.\",\n      \"method\": \"siRNA knockdown of GPC4, Western blot, RT-qPCR, immunofluorescence co-localization, molecular docking\",\n      \"journal\": \"Journal of ethnopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — siRNA KD with defined signaling phenotype, multiple validation methods, single lab\",\n      \"pmids\": [\"41724289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CCN1 (a secreted matricellular protein from radial glial cells) directly interacts with GPC4 on radial glia, and this interaction is required for GPC4 to maintain neural stem cells through the Sonic Hedgehog (Shh) signaling pathway in a heparin-binding-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation/interaction assays, Ccn1 loss-of-function in radial glia, Shh pathway readout, heparin binding assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — interaction and loss-of-function with pathway readout, preprint only\",\n      \"pmids\": [\"bio_10.1101_2025.05.16.654402\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GPC4 co-localizes with Vangl2 presynaptically at mossy fiber boutons in the hippocampus and mediates stabilization of the postsynaptic orphan receptor GPR158; Vangl2-dependent planar cell polarity signaling requires GPC4 to maintain mossy fiber bouton/thorny excrescence synapse morphology and function.\",\n      \"method\": \"Co-localization by immunofluorescence, Vangl2 knockout mouse synapse morphology and electrophysiology\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — co-localization without direct GPC4 manipulation, preprint\",\n      \"pmids\": [\"bio_10.1101_2024.05.28.596141\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"GPC4 is a GPI-anchored heparan sulfate proteoglycan that modulates cell signaling at the cell surface: it acts as a co-receptor in non-canonical Wnt/planar cell polarity signaling (regulating convergent-extension and chondrocyte maturation), is ubiquitinated and degraded by CD36 to suppress β-catenin/c-myc-driven glycolysis, interacts with CCN1 to sustain Shh-dependent neural stem cell maintenance, mediates TLR4/NF-κB inflammatory signaling in glia, and—when shed or upregulated by Aβ in microglia—facilitates tau aggregate seeding and neurodegeneration.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"GPC4 is a GPI-anchored heparan sulfate proteoglycan that functions as a cell-surface co-receptor modulating multiple signaling pathways, including non-canonical Wnt/planar cell polarity, β-catenin, TLR4/NF-κB, and Sonic Hedgehog signaling. In zebrafish, GPC4 is required for non-canonical Wnt signaling during convergent-extension cell movements and chondrocyte maturation in palate morphogenesis and endochondral ossification [PMID:27287801, PMID:27908786]. CD36 promotes proteasome-dependent ubiquitination and degradation of GPC4, thereby suppressing β-catenin/c-myc-driven glycolysis in colorectal cancer [PMID:31484922], while in glial cells GPC4 activates TLR4/NF-κB inflammatory signaling [PMID:37492748, PMID:41724289], and Aβ-induced shedding of GPC4 from microglia facilitates tau aggregate seeding in neurons [PMID:40883746]. Loss-of-function mutations in GPC4 that eliminate the GPI anchor and key glycosylation sites cause Keipert syndrome, a craniofacial-digital disorder recapitulated by Gpc4 knockout mice [PMID:30982611].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing GPC4's molecular identity as a GPI-anchored heparan sulfate proteoglycan resolved what class of molecule GPC4 encodes and how it is tethered to the cell surface.\",\n      \"evidence\": \"cDNA transfection in MDCK cells with biochemical characterization of GPI anchor and HS attachment sites\",\n      \"pmids\": [\"7657705\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No signaling function yet assigned\", \"No in vivo requirement demonstrated\", \"Heparan sulfate chain composition and length uncharacterized\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstration that a conserved tryptophan residue is required for glypican processing and surface HS presentation revealed a structural requirement shared across the glypican family including GPC4.\",\n      \"evidence\": \"Recombinant GPC3 W296R mutant expression and surface HS functional assay, with conservation-based inference to GPC4\",\n      \"pmids\": [\"10814714\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mutagenesis of GPC4 at this residue not performed\", \"Processing mechanism (protease identity, ER/Golgi steps) undefined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Genetic epistasis in zebrafish revealed that GPC4 functions specifically in non-canonical Wnt signaling to drive convergent-extension cell intercalation in chondrocytes and to regulate the timing of endochondral ossification, establishing GPC4 as a co-receptor in PCP signaling.\",\n      \"evidence\": \"Zebrafish gpc4 mutant analysis with epistasis to wnt5b, wnt9a, wls, and frzb mutants across two independent studies\",\n      \"pmids\": [\"27287801\", \"27908786\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical interaction between GPC4 and Wnt ligands not shown\", \"Whether heparan sulfate chains are essential for PCP signal transduction not tested\", \"Mammalian in vivo PCP role not directly demonstrated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identification of CD36 as a GPC4 interactor that triggers its proteasomal degradation revealed a mechanism by which GPC4 protein levels are controlled and linked GPC4 abundance to β-catenin/c-myc-driven glycolytic reprogramming in colorectal cancer.\",\n      \"evidence\": \"Reciprocal Co-IP, proteasome inhibitor rescue, knockdown, and in vivo tumor xenograft models\",\n      \"pmids\": [\"31484922\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ubiquitin ligase responsible for GPC4 ubiquitination not identified\", \"Whether GPC4 directly binds β-catenin or acts via an intermediate not resolved\", \"How CD36-GPC4 interaction triggers ubiquitination mechanistically unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Human genetics linked GPC4 loss-of-function truncating mutations to Keipert syndrome and Gpc4 knockout mice recapitulated the craniofacial-digital phenotype, establishing GPC4 as the causal gene for this Mendelian disorder.\",\n      \"evidence\": \"Whole-exome sequencing of affected families, recombinant protein stability assays, Gpc4 KO mouse phenotyping\",\n      \"pmids\": [\"30982611\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which signaling pathway (Wnt, Shh, FGF) is primarily disrupted in Keipert syndrome not determined\", \"Whether partial loss-of-function alleles produce milder phenotypes unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showing that GPC4 activates TLR4/NF-κB inflammatory signaling in microglia, with its own mRNA stability regulated by FTO-mediated m6A demethylation via YTHDF3, connected GPC4 to innate immune activation and revealed an epitranscriptomic layer of GPC4 regulation.\",\n      \"evidence\": \"RNA stability assays, siRNA knockdown, FTO inhibitor treatment, and rescue experiments in microglia during autoimmune uveitis\",\n      \"pmids\": [\"37492748\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether GPC4 directly binds TLR4 or acts through HS-mediated ligand presentation not distinguished\", \"Single-lab finding without independent replication\", \"In vivo validation of m6A-GPC4 axis limited\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Multiple studies converged to show GPC4 functions in neuroinflammation and neurodegeneration: Aβ-induced GPC4 upregulation and shedding from microglia facilitates tau seeding in neurons, and GPC4 knockdown in astrocytes suppresses TLR4/NF-κB signaling in ischemic stroke.\",\n      \"evidence\": \"Microglia surfaceome profiling, tau uptake/seeding assays, Drosophila in vivo amyloidosis model, astrocyte siRNA knockdown with signaling readouts\",\n      \"pmids\": [\"40883746\", \"41724289\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of GPC4 shedding (sheddase identity) not identified\", \"Whether GPC4-mediated tau seeding is HS-dependent not tested\", \"In vivo mammalian validation of GPC4 in tauopathy lacking\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of GPC4 co-receptor function, the identity of the E3 ligase mediating its ubiquitination, whether HS chains are required for each of its signaling roles, and how GPC4 shedding is regulated in neurodegeneration.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal or cryo-EM structure of GPC4 or GPC4–ligand complexes\", \"HS chain requirements not dissected for individual signaling pathways\", \"Sheddase(s) responsible for GPC4 release not identified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 4, 5, 6, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 3, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 4, 5, 6, 9]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6, 9]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 4, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CD36\",\n      \"TLR4\",\n      \"VANGL2\",\n      \"HS3ST1\",\n      \"CCN1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}