{"gene":"GPI","run_date":"2026-06-10T01:55:21","timeline":{"discoveries":[{"year":2004,"finding":"Insulin-like growth factor binding protein-3 (IGFBP-3) physically binds to AMF/PGI (GPI) at the cell membrane of breast cancer cells, as shown by cross-linking of biotinylated IGFBP-3 to intact cells followed by streptavidin pulldown, and by ligand blotting of purified AMF/PGI. Both glycosylated and nonglycosylated IGFBP-3 inhibited the catalytic (isomerase) activity of AMF/PGI in a dose-dependent manner, inhibited AMF/PGI binding to breast cancer cells, and inhibited AMF/PGI-induced cell migration. IGFBP-3 also decreased AMF/PGI phosphorylation and reduced its translocation to the cell membrane.","method":"Cell-surface cross-linking with biotinylated IGFBP-3, streptavidin pulldown, ligand blotting, enzymatic activity assay, cell migration assay","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal binding confirmed by cross-linking and ligand blot, enzymatic inhibition assay, migration assay; single lab with multiple orthogonal methods","pmids":["15059907"],"is_preprint":false},{"year":2018,"finding":"GPI/AMF (glucose-6-phosphate isomerase/autocrine motility factor) is secreted by glioblastoma cells in an autocrine fashion to stimulate cell migration via its receptor AMFR. Hypoxia upregulates both GPI/AMF and AMFR expression as well as GPI/AMF secretion. Knockdown or inhibition of GPI/AMF reduced glioblastoma cell migration but in part stimulated proliferation, indicating that the cytokine function of secreted GPI/AMF drives invasion while suppressing proliferation. In a highly invasive orthotopic glioblastoma model, GPI/AMF knockdown reduced tumor cell invasion.","method":"shRNA knockdown, in vitro migration assay, orthotopic xenograft model, ELISA for secreted GPI/AMF, receptor expression analysis","journal":"Neuro-oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined cellular phenotype (migration vs. proliferation), in vivo model; single lab with multiple orthogonal methods","pmids":["30053149"],"is_preprint":false},{"year":2014,"finding":"Secreted GPI/AMF acts as a cytokine that promotes epithelial-mesenchymal transition (EMT) and invasiveness in breast cancer multicellular tumor spheroids. Addition of GPI/AMF inhibitors (erythrose-4-phosphate or 6-phosphogluconate) at micromolar doses significantly decreased extracellular GPI/AMF activity (>80%), with concomitant reduction in EMT protein content and migratory capacity, without inhibiting lactate production, indicating that the motogenic effect of GPI/AMF is separable from its glycolytic enzymatic function.","method":"Pharmacological inhibition with erythrose-4-phosphate and 6-phosphogluconate, cell migration assay, EMT protein quantification, extracellular GPI/AMF activity assay, multicellular tumor spheroid model","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition with multiple inhibitors, functional readouts separating enzymatic from cytokine activity; single lab","pmids":["24440856"],"is_preprint":false},{"year":2015,"finding":"AMF/PGI (GPI) promotes endometrial carcinoma cell invasion, migration, and proliferation through its cytokine (not enzymatic) function, acting via the MAPK-ERK1/2 signaling pathway. shRNA-mediated AMF knockdown inhibited these tumorigenic activities, and MEK inhibitor decreased AMF-induced effects. In mouse tumor metastasis models, AMF-silenced cells showed decreased proliferative and metastatic capacity.","method":"shRNA knockdown, MEK inhibitor treatment, 3D culture growth assay, mouse metastasis models (intracardiac and intraperitoneal injection), invasion/migration assays","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined pathway placement (MAPK-ERK), in vivo validation; single lab with multiple orthogonal methods","pmids":["26308071"],"is_preprint":false},{"year":2002,"finding":"Anti-HER2 antibody Herceptin (trastuzumab) downregulates AMF/GPI mRNA and protein expression in high HER2-expressing breast cancer cells, and inhibits transcription from the AMF promoter. HER2 signaling via heregulin (HRG) induces AMF expression, and HCT blocks this induction. Combining HCT with AMF inhibitors (erythrose-4-phosphate or D-mannose-6-phosphate) produced additive inhibitory effects on cell growth and invasiveness, placing GPI/AMF downstream of HER2/heregulin signaling.","method":"RT-PCR, Western blot, promoter-reporter assay, cell proliferation and invasion assays, pharmacological combination treatment","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter reporter assay establishes transcriptional regulation, functional invasion assays; single lab with multiple methods","pmids":["12374700"],"is_preprint":false},{"year":2020,"finding":"Esculetin binds directly to glucose-6-phosphate isomerase (GPI) protein, as demonstrated by microscale thermophoresis (MST). Esculetin inhibited the rate of glycolysis in HepG2 cells, reducing cellular lactate production and glucose consumption. GPD2 was found to bind directly to GPI, revealing a physical interaction between these two glycolysis-related proteins.","method":"Microscale thermophoresis (MST) binding assay, glycolysis rate measurement (lactate production, glucose consumption), protein-protein interaction assay, animal tumor models","journal":"Frontiers in pharmacology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — direct binding shown by MST (single method), functional glycolysis inhibition shown but mechanism not fully resolved; single lab","pmids":["32292350"],"is_preprint":false},{"year":2002,"finding":"AMF receptor (AMFR/gp78) expression in human melanoma cell lines correlates with in vivo metastatic potential. Activation of the AMF receptor in melanoma cells upregulates β3 integrin while downregulating β1 integrin at the cell surface, inducing an integrin phenotype characteristic of invasive/metastatic melanoma, without a mitogenic response.","method":"In vitro AMF stimulation, flow cytometry for integrin expression, in vivo spontaneous metastasis models (immunosuppressed newborn rat, SCID mice), immunohistochemistry","journal":"Clinical & experimental metastasis","confidence":"Low","confidence_rationale":"Tier 3 / Weak — integrin switching demonstrated by flow cytometry after AMF stimulation; single lab, limited mechanistic depth","pmids":["12067203"],"is_preprint":false}],"current_model":"GPI (glucose-6-phosphate isomerase) functions both as an intracellular glycolytic enzyme and as a secreted cytokine (autocrine motility factor, AMF) that binds its receptor AMFR/gp78 to promote tumor cell migration and invasion via MAPK-ERK1/2 signaling; its cytokine activity is separable from its enzymatic activity, is positively regulated downstream of HER2/heregulin signaling, is inhibited by IGFBP-3 binding, and drives EMT and integrin switching in cancer cells."},"narrative":{"mechanistic_narrative":"GPI is a bifunctional protein that operates both as a glycolytic enzyme (glucose-6-phosphate isomerase) and as a secreted cytokine, autocrine motility factor (AMF/PGI), that drives tumor cell migration, invasion, and metastasis [PMID:30053149, PMID:26308071]. Its motogenic cytokine activity is mechanistically separable from its catalytic isomerase activity: pharmacological inhibitors reduce extracellular GPI/AMF activity, EMT marker content, and migration without affecting lactate production [PMID:24440856]. Secreted GPI/AMF signals through its receptor AMFR/gp78 and acts via the MAPK-ERK1/2 cascade to promote invasion, migration, and proliferation, with hypoxia upregulating both ligand and receptor [PMID:30053149, PMID:26308071]. Engagement of AMFR additionally triggers an integrin switch, upregulating β3 and downregulating β1 integrin to confer an invasive phenotype [PMID:12067203]. GPI/AMF expression is positioned downstream of HER2/heregulin signaling, being transcriptionally induced by heregulin and suppressed by anti-HER2 trastuzumab [PMID:12374700], while its cytokine and enzymatic functions are both inhibited by IGFBP-3, which binds GPI/AMF at the cell surface and blocks its phosphorylation, membrane translocation, and migration-promoting activity [PMID:15059907]. Direct physical interaction with the glycolysis-related protein GPD2 has also been detected [PMID:32292350].","teleology":[{"year":2002,"claim":"Established that GPI/AMF expression is governed by upstream oncogenic signaling, placing the cytokine downstream of the HER2/heregulin axis and rationalizing its co-targeting in breast cancer.","evidence":"RT-PCR, Western blot, AMF promoter-reporter assay, and combination inhibitor treatment in HER2-high breast cancer cells","pmids":["12374700"],"confidence":"Medium","gaps":["Transcription factor mediating heregulin-driven AMF induction not identified","Does not address how secreted AMF feeds back on HER2 signaling"]},{"year":2002,"claim":"Linked AMFR activation to a downstream cell-surface consequence — integrin switching — showing how the AMF-receptor axis remodels adhesion toward an invasive phenotype rather than driving proliferation.","evidence":"AMF stimulation with flow cytometry for β1/β3 integrins and in vivo spontaneous metastasis models in melanoma cells","pmids":["12067203"],"confidence":"Low","gaps":["Integrin switching shown by flow cytometry only; signaling intermediates between AMFR and integrin regulation not defined","No direct demonstration that integrin switch is required for metastasis"]},{"year":2004,"claim":"Identified IGFBP-3 as a physical negative regulator of GPI/AMF, establishing that an extracellular binding protein can simultaneously block both its enzymatic and motogenic activities.","evidence":"Cell-surface cross-linking of biotinylated IGFBP-3, streptavidin pulldown, ligand blotting, isomerase activity assay, and migration assay in breast cancer cells","pmids":["15059907"],"confidence":"Medium","gaps":["Binding interface and stoichiometry of the IGFBP-3/GPI interaction unresolved","Mechanism by which IGFBP-3 reduces AMF phosphorylation not defined"]},{"year":2014,"claim":"Demonstrated that the motogenic and glycolytic functions of GPI/AMF are mechanistically separable, showing inhibitors can suppress migration and EMT without affecting glycolytic output.","evidence":"Pharmacological inhibition with erythrose-4-phosphate and 6-phosphogluconate, EMT protein quantification, and migration assays in breast cancer tumor spheroids","pmids":["24440856"],"confidence":"Medium","gaps":["Molecular basis distinguishing cytokine from catalytic activity not structurally defined","EMT transcriptional program downstream of AMF not mapped"]},{"year":2015,"claim":"Placed the cytokine function of GPI/AMF in a defined signaling pathway, showing it drives endometrial carcinoma invasion and metastasis through MAPK-ERK1/2.","evidence":"shRNA knockdown, MEK inhibitor treatment, 3D culture, and mouse metastasis models in endometrial carcinoma","pmids":["26308071"],"confidence":"Medium","gaps":["Receptor-proximal events coupling AMFR to ERK activation not resolved","Does not address tissue-specificity of the proliferation versus invasion balance"]},{"year":2018,"claim":"Showed hypoxia coordinately upregulates GPI/AMF and AMFR and that secreted GPI/AMF drives invasion while suppressing proliferation, distinguishing its autocrine cytokine role from its metabolic role.","evidence":"shRNA knockdown, ELISA for secreted GPI/AMF, migration assays, and orthotopic glioblastoma xenografts","pmids":["30053149"],"confidence":"Medium","gaps":["Mechanism of hypoxia-driven secretion not defined","Molecular basis of the migration/proliferation trade-off unresolved"]},{"year":2020,"claim":"Identified a direct protein-protein interaction between GPI and the glycolysis-related protein GPD2 and showed small-molecule (esculetin) binding can suppress GPI-dependent glycolysis.","evidence":"Microscale thermophoresis binding assays, glycolysis rate measurement, and tumor models in HepG2 cells","pmids":["32292350"],"confidence":"Low","gaps":["GPI-GPD2 interaction shown by single binding method without reciprocal validation","Functional consequence of GPI-GPD2 binding not established"]},{"year":null,"claim":"How the receptor-proximal AMFR signaling complex couples to ERK and integrin remodeling, and what structural features separate GPI's cytokine from its enzymatic activity, remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model distinguishing the catalytic active site from the motogenic surface","AMFR signaling intermediates upstream of ERK and integrin switching not mapped","Regulation of GPI/AMF secretion not mechanistically defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016853","term_label":"isomerase activity","supporting_discovery_ids":[0,2]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[1,3]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[3,6]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[1,2]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,6]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,6]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[2,5]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,3]}],"complexes":[],"partners":["AMFR","IGFBP3","GPD2","ERBB2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P06744","full_name":"Glucose-6-phosphate isomerase","aliases":["Autocrine motility factor","AMF","D-hexose-6-phosphate anomerase","Glucose-6-phosphate C2-epimerase","Neuroleukin","NLK","Phosphoglucose isomerase","PGI","Phosphohexose isomerase","PHI","Sperm antigen 36","SA-36"],"length_aa":558,"mass_kda":63.1,"function":"Isomerase that catalyzes the conversion of alpha-D-glucose-6-phosphate to beta-D-fructose-6-phosphate, the second step in glycolysis, and the reverse reaction in gluconeogenesis, within the cytoplasm (PubMed:28803808). Also shows C2-epimerase activity, interconverting D-glucose-6-phosphate (G6P) and D-mannose-6-phosphate (M6P) (By similarity). Also displays anomerase activity, interconverting alpha and beta-anomeric forms of G6P, D-fructose-6-phosphate and M6P (By similarity). In addition to its metabolic role, this enzyme functions extracellularly as a cytokine: acts as autocrine motility factor (AMF), a secreted angiogenic factor that enhances endothelial cell motility (PubMed:11437381). Functions as neuroleukin, a neurotrophic factor supporting the survival of spinal and sensory neurons (PubMed:11004567, PubMed:3352745). Released by lectin-stimulated T-cells to induce immunoglobulin secretion (PubMed:11004567, PubMed:3352745)","subcellular_location":"Cytoplasm; Secreted","url":"https://www.uniprot.org/uniprotkb/P06744/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/GPI","classification":"Not Classified","n_dependent_lines":547,"n_total_lines":1208,"dependency_fraction":0.45281456953642385},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PSMD1","stoichiometry":10.0},{"gene":"CAPZB","stoichiometry":0.2},{"gene":"ITPR3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/GPI","total_profiled":1310},"omim":[{"mim_id":"621413","title":"PHOSPHOLIPASE A2 INHIBITOR AND LY6/PLAUR DOMAIN-CONTAINING PROTEIN; PINLYP","url":"https://www.omim.org/entry/621413"},{"mim_id":"620781","title":"TRANSMEMBRANE PROTEIN 208; TMEM208","url":"https://www.omim.org/entry/620781"},{"mim_id":"620264","title":"POST-GPI ATTACHMENT TO PROTEINS, GalNAc TRANSFERASE, 4; PGAP4","url":"https://www.omim.org/entry/620264"},{"mim_id":"619985","title":"GLYCOSYLPHOSPHATIDYLINOSITOL BIOSYNTHESIS DEFECT 25; GPIBD25","url":"https://www.omim.org/entry/619985"},{"mim_id":"619979","title":"CHROMOSOME 18 OPEN READING FRAME 32; C18ORF32","url":"https://www.omim.org/entry/619979"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/GPI"},"hgnc":{"alias_symbol":["AMF","NLK"],"prev_symbol":[]},"alphafold":{"accession":"P06744","domains":[{"cath_id":"3.40.50.10490","chopping":"9-76_311-412_474-512","consensus_level":"high","plddt":98.5845,"start":9,"end":512},{"cath_id":"3.40.50.10490","chopping":"101-294","consensus_level":"high","plddt":97.8592,"start":101,"end":294},{"cath_id":"1.10.1390.10","chopping":"517-554","consensus_level":"high","plddt":96.4979,"start":517,"end":554}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P06744","model_url":"https://alphafold.ebi.ac.uk/files/AF-P06744-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P06744-F1-predicted_aligned_error_v6.png","plddt_mean":97.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GPI","jax_strain_url":"https://www.jax.org/strain/search?query=GPI"},"sequence":{"accession":"P06744","fasta_url":"https://rest.uniprot.org/uniprotkb/P06744.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P06744/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P06744"}},"corpus_meta":[{"pmid":"9192898","id":"PMC_9192898","title":"A GPI-linked protein that interacts with Ret to form a candidate neurturin receptor.","date":"1997","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/9192898","citation_count":344,"is_preprint":false},{"pmid":"15040444","id":"PMC_15040444","title":"Sorting GPI-anchored proteins.","date":"2004","source":"Nature reviews. 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II. GPI-anchored and transmembrane molecules internalize through distinct pathways.","date":"1992","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/1346109","citation_count":55,"is_preprint":false},{"pmid":"9193677","id":"PMC_9193677","title":"Cell surface dynamics of GPI-anchored proteins.","date":"1997","source":"Advances in experimental medicine and biology","url":"https://pubmed.ncbi.nlm.nih.gov/9193677","citation_count":54,"is_preprint":false},{"pmid":"23978072","id":"PMC_23978072","title":"GPI transamidase and GPI anchored proteins: oncogenes and biomarkers for cancer.","date":"2013","source":"Critical reviews in biochemistry and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/23978072","citation_count":53,"is_preprint":false},{"pmid":"35015055","id":"PMC_35015055","title":"Vps13-like proteins provide phosphatidylethanolamine for GPI anchor synthesis in the ER.","date":"2022","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/35015055","citation_count":53,"is_preprint":false},{"pmid":"18487995","id":"PMC_18487995","title":"Profiling the expression pattern of GPI transamidase complex subunits in human cancer.","date":"2008","source":"Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc","url":"https://pubmed.ncbi.nlm.nih.gov/18487995","citation_count":51,"is_preprint":false},{"pmid":"31094677","id":"PMC_31094677","title":"Misfolded GPI-anchored proteins are escorted through the secretory pathway by ER-derived factors.","date":"2019","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/31094677","citation_count":49,"is_preprint":false},{"pmid":"16204225","id":"PMC_16204225","title":"Expression of autocrine motility factor (AMF) and its receptor, AMFR, in human breast cancer.","date":"2005","source":"The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society","url":"https://pubmed.ncbi.nlm.nih.gov/16204225","citation_count":48,"is_preprint":false},{"pmid":"27559035","id":"PMC_27559035","title":"Glycosylphosphatidylinositol (GPI) Modification Serves as a Primary Plasmodesmal Sorting Signal.","date":"2016","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/27559035","citation_count":48,"is_preprint":false},{"pmid":"25164783","id":"PMC_25164783","title":"Human genetic disorders involving glycosylphosphatidylinositol (GPI) anchors and glycosphingolipids (GSL).","date":"2014","source":"Journal of inherited metabolic disease","url":"https://pubmed.ncbi.nlm.nih.gov/25164783","citation_count":45,"is_preprint":false},{"pmid":"16291721","id":"PMC_16291721","title":"GPI valence and the fate of secretory membrane proteins in African trypanosomes.","date":"2005","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/16291721","citation_count":45,"is_preprint":false},{"pmid":"29484956","id":"PMC_29484956","title":"Targeting the GPI biosynthetic pathway.","date":"2018","source":"Pathogens and global health","url":"https://pubmed.ncbi.nlm.nih.gov/29484956","citation_count":44,"is_preprint":false},{"pmid":"19703450","id":"PMC_19703450","title":"GPI-anchored proteins at the node of Ranvier.","date":"2009","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/19703450","citation_count":42,"is_preprint":false},{"pmid":"27325793","id":"PMC_27325793","title":"Limited ER quality control for GPI-anchored proteins.","date":"2016","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/27325793","citation_count":42,"is_preprint":false},{"pmid":"8025953","id":"PMC_8025953","title":"Cellular expression of a GPI-linked T cell activation protein.","date":"1994","source":"Cellular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/8025953","citation_count":41,"is_preprint":false},{"pmid":"34162996","id":"PMC_34162996","title":"A knockout cell library of GPI biosynthetic genes for functional studies of GPI-anchored proteins.","date":"2021","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/34162996","citation_count":39,"is_preprint":false},{"pmid":"24128440","id":"PMC_24128440","title":"Recent progress in synthetic and biological studies of GPI anchors and GPI-anchored proteins.","date":"2013","source":"Current opinion in chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/24128440","citation_count":39,"is_preprint":false},{"pmid":"32292350","id":"PMC_32292350","title":"Esculetin Inhibits Cancer Cell Glycolysis by Binding Tumor PGK2, GPD2, and GPI.","date":"2020","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/32292350","citation_count":39,"is_preprint":false},{"pmid":"32573677","id":"PMC_32573677","title":"Mammalian GPI-anchor modifications and the enzymes involved.","date":"2020","source":"Biochemical Society transactions","url":"https://pubmed.ncbi.nlm.nih.gov/32573677","citation_count":36,"is_preprint":false},{"pmid":"27760325","id":"PMC_27760325","title":"HSV1 MicroRNA Modulation of GPI Anchoring and Downstream Immune Evasion.","date":"2016","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/27760325","citation_count":36,"is_preprint":false},{"pmid":"32828789","id":"PMC_32828789","title":"Exosomes and GPI-anchored proteins: Judicious pairs for investigating biomarkers from body fluids.","date":"2020","source":"Advanced drug delivery reviews","url":"https://pubmed.ncbi.nlm.nih.gov/32828789","citation_count":35,"is_preprint":false},{"pmid":"15376625","id":"PMC_15376625","title":"GPI-anchored protein cleavage in the regulation of transmembrane signals.","date":"2004","source":"Sub-cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15376625","citation_count":34,"is_preprint":false},{"pmid":"32418035","id":"PMC_32418035","title":"GPI Anchored Proteins in Aspergillus fumigatus and Cell Wall Morphogenesis.","date":"2020","source":"Current topics in microbiology and immunology","url":"https://pubmed.ncbi.nlm.nih.gov/32418035","citation_count":33,"is_preprint":false},{"pmid":"26015286","id":"PMC_26015286","title":"Trafficking and Membrane Organization of GPI-Anchored Proteins in Health and Diseases.","date":"2015","source":"Current topics in membranes","url":"https://pubmed.ncbi.nlm.nih.gov/26015286","citation_count":33,"is_preprint":false},{"pmid":"29602703","id":"PMC_29602703","title":"Pertussis in Africa: Findings and recommendations of the Global Pertussis Initiative (GPI).","date":"2018","source":"Vaccine","url":"https://pubmed.ncbi.nlm.nih.gov/29602703","citation_count":33,"is_preprint":false},{"pmid":"17113867","id":"PMC_17113867","title":"CHO glycosylation mutants: GPI anchor.","date":"2006","source":"Methods in enzymology","url":"https://pubmed.ncbi.nlm.nih.gov/17113867","citation_count":32,"is_preprint":false},{"pmid":"15646015","id":"PMC_15646015","title":"GPI-anchored proteins and lipid rafts.","date":"2004","source":"The Italian journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15646015","citation_count":32,"is_preprint":false},{"pmid":"15059907","id":"PMC_15059907","title":"Insulin-like growth factor binding protein-3 interacts with autocrine motility factor/phosphoglucose isomerase (AMF/PGI) and inhibits the AMF/PGI function.","date":"2004","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/15059907","citation_count":31,"is_preprint":false},{"pmid":"31715252","id":"PMC_31715252","title":"Novel strategy for anchorage position control of GPI-attached proteins in the yeast cell wall using different GPI-anchoring domains.","date":"2019","source":"Metabolic engineering","url":"https://pubmed.ncbi.nlm.nih.gov/31715252","citation_count":31,"is_preprint":false},{"pmid":"30684517","id":"PMC_30684517","title":"Vaccination against babesiosis using recombinant GPI-anchored proteins.","date":"2019","source":"International journal for parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/30684517","citation_count":30,"is_preprint":false},{"pmid":"18058122","id":"PMC_18058122","title":"Characterizing the interactions between GPI-anchored alkaline phosphatases and membrane domains by AFM.","date":"2007","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/18058122","citation_count":28,"is_preprint":false},{"pmid":"28734155","id":"PMC_28734155","title":"Activation of mTOR is involved in anti-β2GPI/β2GPI-induced expression of tissue factor and IL-8 in monocytes.","date":"2017","source":"Thrombosis research","url":"https://pubmed.ncbi.nlm.nih.gov/28734155","citation_count":28,"is_preprint":false},{"pmid":"15470101","id":"PMC_15470101","title":"Yeast Kre1p is GPI-anchored and involved in both cell wall assembly and architecture.","date":"2004","source":"Microbiology (Reading, England)","url":"https://pubmed.ncbi.nlm.nih.gov/15470101","citation_count":27,"is_preprint":false},{"pmid":"22411310","id":"PMC_22411310","title":"Lipid rafts, caveolae and GPI-linked proteins.","date":"2012","source":"Advances in experimental medicine and biology","url":"https://pubmed.ncbi.nlm.nih.gov/22411310","citation_count":26,"is_preprint":false},{"pmid":"30053149","id":"PMC_30053149","title":"The secreted glycolytic enzyme GPI/AMF stimulates glioblastoma cell migration and invasion in an autocrine fashion but can have anti-proliferative effects.","date":"2018","source":"Neuro-oncology","url":"https://pubmed.ncbi.nlm.nih.gov/30053149","citation_count":26,"is_preprint":false},{"pmid":"11532956","id":"PMC_11532956","title":"Purification, cloning and characterization of a GPI inositol deacylase from Trypanosoma brucei.","date":"2001","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/11532956","citation_count":26,"is_preprint":false},{"pmid":"31319476","id":"PMC_31319476","title":"Endoplasmic Reticulum Export of GPI-Anchored Proteins.","date":"2019","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31319476","citation_count":25,"is_preprint":false},{"pmid":"36828365","id":"PMC_36828365","title":"Accumulated precursors of specific GPI-anchored proteins upregulate GPI biosynthesis with ARV1.","date":"2023","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/36828365","citation_count":25,"is_preprint":false},{"pmid":"37742547","id":"PMC_37742547","title":"Effects of static magnetic field (SMF) and alternating magnetic field (AMF) assisted freezing on the microstructure and protein properties of channel catfish (Ictalurus punctatus) fillet.","date":"2023","source":"Food chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37742547","citation_count":25,"is_preprint":false},{"pmid":"31739586","id":"PMC_31739586","title":"Relating GPI-Anchored Ly6 Proteins uPAR and CD59 to Viral Infection.","date":"2019","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/31739586","citation_count":24,"is_preprint":false},{"pmid":"37684232","id":"PMC_37684232","title":"Structures of liganded glycosylphosphatidylinositol transamidase illuminate GPI-AP biogenesis.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/37684232","citation_count":24,"is_preprint":false},{"pmid":"24440856","id":"PMC_24440856","title":"GPI/AMF inhibition blocks the development of the metastatic phenotype of mature multi-cellular tumor spheroids.","date":"2014","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/24440856","citation_count":24,"is_preprint":false},{"pmid":"32967966","id":"PMC_32967966","title":"Calnexin mediates the maturation of GPI-anchors through ER retention.","date":"2020","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/32967966","citation_count":23,"is_preprint":false},{"pmid":"10820431","id":"PMC_10820431","title":"Expression of the AMF/neuroleukin receptor in developing and adult brain cerebellum.","date":"2000","source":"Journal of neuroscience research","url":"https://pubmed.ncbi.nlm.nih.gov/10820431","citation_count":23,"is_preprint":false},{"pmid":"28459879","id":"PMC_28459879","title":"Controlling transferrin receptor trafficking with GPI-valence in bloodstream stage African trypanosomes.","date":"2017","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/28459879","citation_count":23,"is_preprint":false},{"pmid":"25970100","id":"PMC_25970100","title":"Both PIGA and PIGL mutations cause GPI-a deficient isolates in the Tk6 cell line.","date":"2015","source":"Environmental and molecular mutagenesis","url":"https://pubmed.ncbi.nlm.nih.gov/25970100","citation_count":23,"is_preprint":false},{"pmid":"19724897","id":"PMC_19724897","title":"Annexin A2 mediates anti-beta 2 GPI/beta 2 GPI-induced tissue factor expression on monocytes.","date":"2009","source":"International journal of molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/19724897","citation_count":23,"is_preprint":false},{"pmid":"33035180","id":"PMC_33035180","title":"Diversity of GPI-anchored fungal adhesins.","date":"2020","source":"Biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/33035180","citation_count":22,"is_preprint":false},{"pmid":"21448495","id":"PMC_21448495","title":"Synthetic glycosylphosphatidylinositol (GPI) anchors: how these complex molecules have been made.","date":"2011","source":"Natural product reports","url":"https://pubmed.ncbi.nlm.nih.gov/21448495","citation_count":22,"is_preprint":false},{"pmid":"25948169","id":"PMC_25948169","title":"Each GPI-anchored protein species forms a specific lipid raft depending on its GPI attachment signal.","date":"2015","source":"Glycoconjugate journal","url":"https://pubmed.ncbi.nlm.nih.gov/25948169","citation_count":22,"is_preprint":false},{"pmid":"20057357","id":"PMC_20057357","title":"GPI anchoring facilitates propagation and spread of misfolded Sup35 aggregates in mammalian cells.","date":"2010","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/20057357","citation_count":22,"is_preprint":false},{"pmid":"21660958","id":"PMC_21660958","title":"Effects of membrane cholesterol depletion and GPI-anchored protein reduction on osteoblastic mechanotransduction.","date":"2011","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/21660958","citation_count":21,"is_preprint":false},{"pmid":"18445040","id":"PMC_18445040","title":"Hypoxia induces expression of a GPI-anchorless splice variant of the prion protein.","date":"2008","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/18445040","citation_count":20,"is_preprint":false},{"pmid":"36550078","id":"PMC_36550078","title":"Platelet-derived microparticles stimulated by anti-β2GPI/β2GPI complexes induce pyroptosis of endothelial cells in antiphospholipid syndrome.","date":"2023","source":"Platelets","url":"https://pubmed.ncbi.nlm.nih.gov/36550078","citation_count":20,"is_preprint":false},{"pmid":"7531502","id":"PMC_7531502","title":"Purified GPI-anchored CD4DAF as a receptor for HIV-mediated gene transfer.","date":"1994","source":"Human gene therapy","url":"https://pubmed.ncbi.nlm.nih.gov/7531502","citation_count":20,"is_preprint":false},{"pmid":"12067203","id":"PMC_12067203","title":"Expression and function of the AMF receptor by human melanoma in experimental and clinical systems.","date":"2002","source":"Clinical & experimental metastasis","url":"https://pubmed.ncbi.nlm.nih.gov/12067203","citation_count":19,"is_preprint":false},{"pmid":"12374700","id":"PMC_12374700","title":"Antihuman epidermal growth factor receptor 2 antibody herceptin inhibits autocrine motility factor (AMF) expression and potentiates antitumor effects of AMF inhibitors.","date":"2002","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/12374700","citation_count":19,"is_preprint":false},{"pmid":"35316654","id":"PMC_35316654","title":"Self-incompatibility requires GPI anchor remodeling by the poppy PGAP1 ortholog HLD1.","date":"2022","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/35316654","citation_count":18,"is_preprint":false},{"pmid":"18028549","id":"PMC_18028549","title":"Alterations of GPI transamidase subunits in head and neck squamous carcinoma.","date":"2007","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/18028549","citation_count":18,"is_preprint":false},{"pmid":"33838053","id":"PMC_33838053","title":"Human SND2 mediates ER targeting of GPI-anchored proteins with low hydrophobic GPI attachment signals.","date":"2021","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/33838053","citation_count":18,"is_preprint":false},{"pmid":"23990786","id":"PMC_23990786","title":"Determinants of GPI-PLC localisation to the flagellum and access to GPI-anchored substrates in trypanosomes.","date":"2013","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/23990786","citation_count":17,"is_preprint":false},{"pmid":"32165008","id":"PMC_32165008","title":"Homozygous splice-variants in human ARV1 cause GPI-anchor synthesis deficiency.","date":"2020","source":"Molecular genetics and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/32165008","citation_count":17,"is_preprint":false},{"pmid":"26308071","id":"PMC_26308071","title":"AMF/PGI-mediated tumorigenesis through MAPK-ERK signaling in endometrial carcinoma.","date":"2015","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/26308071","citation_count":16,"is_preprint":false},{"pmid":"34618080","id":"PMC_34618080","title":"AtPGAP1 functions as a GPI inositol-deacylase required for efficient transport of GPI-anchored proteins.","date":"2021","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/34618080","citation_count":16,"is_preprint":false},{"pmid":"15275478","id":"PMC_15275478","title":"GPI-anchor synthesis.","date":"1994","source":"Parasitology today (Personal ed.)","url":"https://pubmed.ncbi.nlm.nih.gov/15275478","citation_count":15,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":43059,"output_tokens":2596,"usd":0.084058,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9252,"output_tokens":2813,"usd":0.058292,"stage2_stop_reason":"end_turn"},"total_usd":0.14235,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"Looking at the corpus, I need to identify papers about the canonical GPI gene (glucose-6-phosphate isomerase / autocrine motility factor, AMF). Many papers in this corpus are about GPI-anchored proteins generally (a post-translational modification), not the GPI gene itself. I need to carefully distinguish:\n\n1. Papers about GPI/AMF (glucose-6-phosphate isomerase / autocrine motility factor) - the specific enzyme encoded by the GPI gene\n2. Papers about GPI-anchored proteins generally - these are about the modification, not the GPI gene\n\nRelevant papers about the GPI gene (glucose-6-phosphate isomerase/AMF):\n- PMID 15059907: IGFBP-3 binds AMF/PGI and inhibits its function\n- PMID 30053149: GPI/AMF role in glioblastoma migration\n- PMID 24440856: GPI/AMF inhibition in metastatic phenotype\n- PMID 26308071: AMF/PGI-mediated tumorigenesis via MAPK-ERK\n- PMID 12374700: HER2 antibody inhibits AMF expression\n- PMID 32292350: Esculetin binds GPI (glucose-6-phosphate isomerase) and inhibits glycolysis\n- PMID 10846067: AMF-1/Gps2 (this is a different protein - Gps2, not GPI/AMF)\n- PMID 16204225: AMF/AMFR expression in breast cancer - mostly correlative\n- PMID 10820431: AMF receptor expression in cerebellum\n- PMID 12067203: AMF receptor in melanoma\n\nLet me now extract only mechanistic discoveries:\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"Insulin-like growth factor binding protein-3 (IGFBP-3) physically binds to AMF/PGI (GPI) at the cell membrane of breast cancer cells, as shown by cross-linking of biotinylated IGFBP-3 to intact cells followed by streptavidin pulldown, and by ligand blotting of purified AMF/PGI. Both glycosylated and nonglycosylated IGFBP-3 inhibited the catalytic (isomerase) activity of AMF/PGI in a dose-dependent manner, inhibited AMF/PGI binding to breast cancer cells, and inhibited AMF/PGI-induced cell migration. IGFBP-3 also decreased AMF/PGI phosphorylation and reduced its translocation to the cell membrane.\",\n      \"method\": \"Cell-surface cross-linking with biotinylated IGFBP-3, streptavidin pulldown, ligand blotting, enzymatic activity assay, cell migration assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding confirmed by cross-linking and ligand blot, enzymatic inhibition assay, migration assay; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"15059907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"GPI/AMF (glucose-6-phosphate isomerase/autocrine motility factor) is secreted by glioblastoma cells in an autocrine fashion to stimulate cell migration via its receptor AMFR. Hypoxia upregulates both GPI/AMF and AMFR expression as well as GPI/AMF secretion. Knockdown or inhibition of GPI/AMF reduced glioblastoma cell migration but in part stimulated proliferation, indicating that the cytokine function of secreted GPI/AMF drives invasion while suppressing proliferation. In a highly invasive orthotopic glioblastoma model, GPI/AMF knockdown reduced tumor cell invasion.\",\n      \"method\": \"shRNA knockdown, in vitro migration assay, orthotopic xenograft model, ELISA for secreted GPI/AMF, receptor expression analysis\",\n      \"journal\": \"Neuro-oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined cellular phenotype (migration vs. proliferation), in vivo model; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"30053149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Secreted GPI/AMF acts as a cytokine that promotes epithelial-mesenchymal transition (EMT) and invasiveness in breast cancer multicellular tumor spheroids. Addition of GPI/AMF inhibitors (erythrose-4-phosphate or 6-phosphogluconate) at micromolar doses significantly decreased extracellular GPI/AMF activity (>80%), with concomitant reduction in EMT protein content and migratory capacity, without inhibiting lactate production, indicating that the motogenic effect of GPI/AMF is separable from its glycolytic enzymatic function.\",\n      \"method\": \"Pharmacological inhibition with erythrose-4-phosphate and 6-phosphogluconate, cell migration assay, EMT protein quantification, extracellular GPI/AMF activity assay, multicellular tumor spheroid model\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition with multiple inhibitors, functional readouts separating enzymatic from cytokine activity; single lab\",\n      \"pmids\": [\"24440856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"AMF/PGI (GPI) promotes endometrial carcinoma cell invasion, migration, and proliferation through its cytokine (not enzymatic) function, acting via the MAPK-ERK1/2 signaling pathway. shRNA-mediated AMF knockdown inhibited these tumorigenic activities, and MEK inhibitor decreased AMF-induced effects. In mouse tumor metastasis models, AMF-silenced cells showed decreased proliferative and metastatic capacity.\",\n      \"method\": \"shRNA knockdown, MEK inhibitor treatment, 3D culture growth assay, mouse metastasis models (intracardiac and intraperitoneal injection), invasion/migration assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined pathway placement (MAPK-ERK), in vivo validation; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"26308071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Anti-HER2 antibody Herceptin (trastuzumab) downregulates AMF/GPI mRNA and protein expression in high HER2-expressing breast cancer cells, and inhibits transcription from the AMF promoter. HER2 signaling via heregulin (HRG) induces AMF expression, and HCT blocks this induction. Combining HCT with AMF inhibitors (erythrose-4-phosphate or D-mannose-6-phosphate) produced additive inhibitory effects on cell growth and invasiveness, placing GPI/AMF downstream of HER2/heregulin signaling.\",\n      \"method\": \"RT-PCR, Western blot, promoter-reporter assay, cell proliferation and invasion assays, pharmacological combination treatment\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter reporter assay establishes transcriptional regulation, functional invasion assays; single lab with multiple methods\",\n      \"pmids\": [\"12374700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Esculetin binds directly to glucose-6-phosphate isomerase (GPI) protein, as demonstrated by microscale thermophoresis (MST). Esculetin inhibited the rate of glycolysis in HepG2 cells, reducing cellular lactate production and glucose consumption. GPD2 was found to bind directly to GPI, revealing a physical interaction between these two glycolysis-related proteins.\",\n      \"method\": \"Microscale thermophoresis (MST) binding assay, glycolysis rate measurement (lactate production, glucose consumption), protein-protein interaction assay, animal tumor models\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — direct binding shown by MST (single method), functional glycolysis inhibition shown but mechanism not fully resolved; single lab\",\n      \"pmids\": [\"32292350\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"AMF receptor (AMFR/gp78) expression in human melanoma cell lines correlates with in vivo metastatic potential. Activation of the AMF receptor in melanoma cells upregulates β3 integrin while downregulating β1 integrin at the cell surface, inducing an integrin phenotype characteristic of invasive/metastatic melanoma, without a mitogenic response.\",\n      \"method\": \"In vitro AMF stimulation, flow cytometry for integrin expression, in vivo spontaneous metastasis models (immunosuppressed newborn rat, SCID mice), immunohistochemistry\",\n      \"journal\": \"Clinical & experimental metastasis\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — integrin switching demonstrated by flow cytometry after AMF stimulation; single lab, limited mechanistic depth\",\n      \"pmids\": [\"12067203\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GPI (glucose-6-phosphate isomerase) functions both as an intracellular glycolytic enzyme and as a secreted cytokine (autocrine motility factor, AMF) that binds its receptor AMFR/gp78 to promote tumor cell migration and invasion via MAPK-ERK1/2 signaling; its cytokine activity is separable from its enzymatic activity, is positively regulated downstream of HER2/heregulin signaling, is inhibited by IGFBP-3 binding, and drives EMT and integrin switching in cancer cells.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GPI is a bifunctional protein that operates both as a glycolytic enzyme (glucose-6-phosphate isomerase) and as a secreted cytokine, autocrine motility factor (AMF/PGI), that drives tumor cell migration, invasion, and metastasis [#1, #3]. Its motogenic cytokine activity is mechanistically separable from its catalytic isomerase activity: pharmacological inhibitors reduce extracellular GPI/AMF activity, EMT marker content, and migration without affecting lactate production [#2]. Secreted GPI/AMF signals through its receptor AMFR/gp78 and acts via the MAPK-ERK1/2 cascade to promote invasion, migration, and proliferation, with hypoxia upregulating both ligand and receptor [#1, #3]. Engagement of AMFR additionally triggers an integrin switch, upregulating β3 and downregulating β1 integrin to confer an invasive phenotype [#6]. GPI/AMF expression is positioned downstream of HER2/heregulin signaling, being transcriptionally induced by heregulin and suppressed by anti-HER2 trastuzumab [#4], while its cytokine and enzymatic functions are both inhibited by IGFBP-3, which binds GPI/AMF at the cell surface and blocks its phosphorylation, membrane translocation, and migration-promoting activity [#0]. Direct physical interaction with the glycolysis-related protein GPD2 has also been detected [#5].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established that GPI/AMF expression is governed by upstream oncogenic signaling, placing the cytokine downstream of the HER2/heregulin axis and rationalizing its co-targeting in breast cancer.\",\n      \"evidence\": \"RT-PCR, Western blot, AMF promoter-reporter assay, and combination inhibitor treatment in HER2-high breast cancer cells\",\n      \"pmids\": [\"12374700\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcription factor mediating heregulin-driven AMF induction not identified\", \"Does not address how secreted AMF feeds back on HER2 signaling\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Linked AMFR activation to a downstream cell-surface consequence — integrin switching — showing how the AMF-receptor axis remodels adhesion toward an invasive phenotype rather than driving proliferation.\",\n      \"evidence\": \"AMF stimulation with flow cytometry for β1/β3 integrins and in vivo spontaneous metastasis models in melanoma cells\",\n      \"pmids\": [\"12067203\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Integrin switching shown by flow cytometry only; signaling intermediates between AMFR and integrin regulation not defined\", \"No direct demonstration that integrin switch is required for metastasis\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified IGFBP-3 as a physical negative regulator of GPI/AMF, establishing that an extracellular binding protein can simultaneously block both its enzymatic and motogenic activities.\",\n      \"evidence\": \"Cell-surface cross-linking of biotinylated IGFBP-3, streptavidin pulldown, ligand blotting, isomerase activity assay, and migration assay in breast cancer cells\",\n      \"pmids\": [\"15059907\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding interface and stoichiometry of the IGFBP-3/GPI interaction unresolved\", \"Mechanism by which IGFBP-3 reduces AMF phosphorylation not defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated that the motogenic and glycolytic functions of GPI/AMF are mechanistically separable, showing inhibitors can suppress migration and EMT without affecting glycolytic output.\",\n      \"evidence\": \"Pharmacological inhibition with erythrose-4-phosphate and 6-phosphogluconate, EMT protein quantification, and migration assays in breast cancer tumor spheroids\",\n      \"pmids\": [\"24440856\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis distinguishing cytokine from catalytic activity not structurally defined\", \"EMT transcriptional program downstream of AMF not mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placed the cytokine function of GPI/AMF in a defined signaling pathway, showing it drives endometrial carcinoma invasion and metastasis through MAPK-ERK1/2.\",\n      \"evidence\": \"shRNA knockdown, MEK inhibitor treatment, 3D culture, and mouse metastasis models in endometrial carcinoma\",\n      \"pmids\": [\"26308071\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor-proximal events coupling AMFR to ERK activation not resolved\", \"Does not address tissue-specificity of the proliferation versus invasion balance\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed hypoxia coordinately upregulates GPI/AMF and AMFR and that secreted GPI/AMF drives invasion while suppressing proliferation, distinguishing its autocrine cytokine role from its metabolic role.\",\n      \"evidence\": \"shRNA knockdown, ELISA for secreted GPI/AMF, migration assays, and orthotopic glioblastoma xenografts\",\n      \"pmids\": [\"30053149\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of hypoxia-driven secretion not defined\", \"Molecular basis of the migration/proliferation trade-off unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified a direct protein-protein interaction between GPI and the glycolysis-related protein GPD2 and showed small-molecule (esculetin) binding can suppress GPI-dependent glycolysis.\",\n      \"evidence\": \"Microscale thermophoresis binding assays, glycolysis rate measurement, and tumor models in HepG2 cells\",\n      \"pmids\": [\"32292350\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"GPI-GPD2 interaction shown by single binding method without reciprocal validation\", \"Functional consequence of GPI-GPD2 binding not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the receptor-proximal AMFR signaling complex couples to ERK and integrin remodeling, and what structural features separate GPI's cytokine from its enzymatic activity, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model distinguishing the catalytic active site from the motogenic surface\", \"AMFR signaling intermediates upstream of ERK and integrin switching not mapped\", \"Regulation of GPI/AMF secretion not mechanistically defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 6]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [2, 5]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"AMFR\", \"IGFBP3\", \"GPD2\", \"ERBB2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}