{"gene":"AMN","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2001,"finding":"AMN encodes a novel type I transmembrane protein expressed exclusively in the extra-embryonic visceral endoderm during mouse gastrulation. The extracellular region contains a cysteine-rich domain with similarity to BMP-binding domains in chordin and Short gastrulation, suggesting AMN may modulate a BMP signaling pathway in the visceral endoderm to direct trunk mesoderm formation from the middle primitive streak.","method":"Gene cloning, transgene-induced insertional mutation, in situ hybridization, domain analysis, mouse embryo phenotyping","journal":"Nature genetics","confidence":"Medium","confidence_rationale":"Tier 2 — direct loss-of-function phenotype with localization, but BMP-binding is inferred from domain homology, not directly demonstrated","pmids":["11279523"],"is_preprint":false},{"year":1998,"finding":"The amn gene product is required in extraembryonic tissues (visceral endoderm) for the generation of middle primitive streak derivatives. Chimera experiments using wild-type ES cells in amnionless-/- blastocysts demonstrated that the amnionless function in visceral endoderm is cell-non-autonomous with respect to the embryo proper, establishing a signaling role for AMN in the visceral endoderm during gastrulation.","method":"ES cell↔blastocyst chimera analysis, histological and molecular phenotyping of amnionless mutant embryos","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis via chimera experiment with defined tissue-specific requirement","pmids":["9851841"],"is_preprint":false},{"year":2003,"finding":"Cubilin and AMN form a tight heteromeric complex (cubam). AMN binds to the amino-terminal third of cubilin, directs cubilin trafficking from early biosynthetic compartments to the cell surface and endosomes, and enables intrinsic factor-cobalamin endocytosis and lysosomal degradation. Neither protein alone confers ligand endocytosis; cotransfection of both is required for functional receptor activity.","method":"Co-purification by IF-cobalamin affinity chromatography and non-denaturing gel filtration; transfection of polarized epithelial cells; colocalization by immunofluorescence; ligand endocytosis and lysosomal degradation assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 — biochemical co-purification plus functional reconstitution in transfected cells with multiple orthogonal readouts","pmids":["14576052"],"is_preprint":false},{"year":2003,"finding":"Homozygous mutations affecting exons 1–4 of human AMN cause selective intestinal malabsorption of vitamin B12 (Imerslund-Gräsbeck syndrome) without affecting embryonic development, indicating the 5′ end of AMN is dispensable for gastrulation but necessary for cobalamin absorption. When the 5′ end is truncated, translation initiates from alternative downstream start codons.","method":"Human genetic mutation analysis, translation initiation site mapping","journal":"Nature genetics","confidence":"Medium","confidence_rationale":"Tier 2 — human loss-of-function genetics with defined molecular consequence (alternative translation initiation)","pmids":["12590260"],"is_preprint":false},{"year":2004,"finding":"AMN is expressed in kidney proximal tubules and intestinal epithelium in addition to visceral endoderm. In Amn−/− ES cell↔wild-type blastocyst chimeras, Cubn is not properly localized to the cell surface in Amn−/− tissues, adult chimeras exhibit selective proteinuria of Cubn ligands, and Amn−/− embryos show failure of epiblast growth linked to middle primitive streak assembly. This establishes AMN as an essential in vivo component of the Cubn receptor complex mediating endocytosis/transcytosis in polarized epithelia.","method":"ES cell↔blastocyst chimera generation, immunolocalization of Cubn in embryo and adult mouse tissues, urinary protein analysis","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 — in vivo genetic loss-of-function with direct protein localization and functional (proteinuria) readout, replicated across tissue types","pmids":["15342463"],"is_preprint":false},{"year":2005,"finding":"In vivo canine models of I-GS with AMN mutations (in-frame deletion in exon 10 or substitution in the translation initiation codon) demonstrate that loss of AMN expression blocks cubilin processing and targeting to the apical brush-border membrane, abolishing functional receptor activity. These features are recapitulated in a heterologous cell-transfection system, validating AMN's essential role in cubilin apical membrane targeting.","method":"Canine genetic model, in vivo tissue studies, heterologous cell transfection, immunohistochemistry","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — in vivo animal model with defined mutations, direct protein localization, replicated in cell-based system","pmids":["15845892"],"is_preprint":false},{"year":2005,"finding":"AMN interacts with the EGF domains of cubilin (not the N-terminal domain) and is responsible for membrane attachment and ER export of the cubilin-AMN complex. Cubilin itself mediates apical sorting in a carbohydrate-dependent manner (sensitive to tunicamycin). Truncated cubilin constructs lacking the N-terminal domain reach the apical membrane only when co-expressed with AMN.","method":"Expression of AMN and cubilin fragments in polarized MDCK cells, co-immunoprecipitation, colocalization, tunicamycin treatment","journal":"Journal of the American Society of Nephrology","confidence":"High","confidence_rationale":"Tier 2 — systematic domain-mapping with co-IP and functional trafficking readout in polarized cells","pmids":["15976000"],"is_preprint":false},{"year":2008,"finding":"In renal brush-border membranes, cubilin exists in a functional complex with both AMN and megalin. Three distinct regions of cubilin (N-terminus, CUB domains 12–17, CUB domains 22–27) show Ca2+-dependent binding to megalin in vitro. Silencing megalin or AMN in opossum kidney cells reduces cubilin levels by 85–90% and decreases cubilin half-life 2-fold, indicating that interactions with both megalin and AMN are required for cubilin intracellular stability.","method":"Ligand-affinity chromatography, immunoprecipitation of renal brush-border membranes, in vitro Ca2+-dependent binding assays, gene silencing (siRNA), turnover/half-life studies, immunohistochemistry","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including in vitro binding, co-IP of native membranes, and functional gene silencing with quantitative protein stability readout","pmids":["17990981"],"is_preprint":false},{"year":2010,"finding":"The cytosolic domain of AMN contains two FXNPXF motifs that are functionally redundant and mediate cubam endocytosis by interacting with the clathrin-associated sorting proteins Dab2 and ARH. Sequential mutagenesis of each motif combined with yeast two-hybrid analysis demonstrated that either signal alone is sufficient to drive endocytosis through Dab2 or ARH.","method":"Sequential mutagenesis of AMN cytoplasmic FXNPXF motifs, expression of AMN mutant panel, yeast two-hybrid interaction assays, functional endocytosis assays","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis combined with yeast two-hybrid and functional endocytosis assay, defining molecular endocytic mechanism","pmids":["20088845"],"is_preprint":false},{"year":2011,"finding":"Compound heterozygous AMN mutations (premature termination codons in exons 6 and 7) dramatically decrease cubilin receptor activity at the luminal surface of intestinal cells in humans, despite normal cubilin affinity for intrinsic factor. This demonstrates that AMN is essential for correct luminal (apical) expression of cubilin in vivo.","method":"Human genetic analysis, measurement of cubilin receptor activity in urine, intrinsic factor binding affinity assay","journal":"Haematologica","confidence":"Medium","confidence_rationale":"Tier 2 — human loss-of-function with direct functional receptor activity measurement, but single case study","pmids":["21750092"],"is_preprint":false},{"year":2012,"finding":"Drosophila orthologs of cubilin (dCubilin) and amnionless (dAMN) are specifically expressed in nephrocytes where they function as co-receptors for protein uptake. Human AMN expressed in Drosophila nephrocytes rescues the protein-uptake defect caused by dAMN knockdown, demonstrating evolutionary conservation of cubilin/AMN co-receptor function. Cubilin/AMN-mediated protein reabsorption is required for maintenance of nephrocyte ultrastructure and fly survival under toxic stress.","method":"Genetic screen, targeted RNAi knockdown in Drosophila nephrocytes, transgenic rescue with human AMN, protein uptake assays, electron microscopy of ultrastructure","journal":"Journal of the American Society of Nephrology","confidence":"High","confidence_rationale":"Tier 2 — cross-species functional rescue experiment with multiple readouts (protein uptake, ultrastructure, survival), confirming conserved mechanism","pmids":["23264686"],"is_preprint":false},{"year":2014,"finding":"In adult human terminal ileum, cubilin and AMN co-localize to epithelial cells but megalin protein is absent (with extremely low LRP2 mRNA). This demonstrates that cubilin and AMN can function independently of megalin for vitamin B12 absorption in the intestine, establishing tissue-specific and ligand-specific dependence on megalin.","method":"Immunohistochemistry, quantitative RT-PCR for LRP2 mRNA in adult human terminal ileum tissue","journal":"Physiological reports","confidence":"Medium","confidence_rationale":"Tier 3 — direct protein localization in human tissue, but mechanistic inference rather than functional experiment","pmids":["25052491"],"is_preprint":false},{"year":2018,"finding":"AMN-mediated glycosylation of cubilin is necessary (not merely AMN-cubilin interaction) for plasma membrane expression of the complex. N-linked glycosylation of at least 4 cubilin residues is required for surface targeting. Pathogenic AMN and CUBN missense mutations cause ER retention and completely block amnionless-dependent plasma membrane expression of cubilin, as confirmed in renal proximal tubular cells from an IGS patient.","method":"Quantitative membrane targeting assay in cultured renal and intestinal cells, quantitative mass spectrometry, site-directed mutagenesis of N-glycosylation sites, patient-derived proximal tubular cell analysis","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis plus mass spectrometry plus patient cell validation, with quantitative functional readout","pmids":["29402915"],"is_preprint":false},{"year":2022,"finding":"In Drosophila nephrocytes, store-operated calcium entry (SOCE) mediated by Stim/Orai regulates the abundance and localization of dAMN (the amnionless ortholog). RNAi knockdown of Stim or Orai reduced dAMN abundance, impaired albumin binding and endocytosis, and disrupted slit diaphragm organization. Calcium chelation (EGTA) reduced albumin binding following pre-treatment, linking SOCE to AMN-dependent endocytic function.","method":"GCaMP6 calcium imaging in vivo and in vitro, RNAi knockdown of SOCE genes (Stim, Orai), pharmacological inhibition (EGTA, 2-APB), albumin uptake assay, immunolocalization of dAMN","journal":"Journal of insect physiology","confidence":"Medium","confidence_rationale":"Tier 2 — genetic knockdown with calcium imaging and functional endocytosis readout in Drosophila ortholog model","pmids":["36341969"],"is_preprint":false},{"year":2025,"finding":"In a Drosophila model of Dent's disease, depletion of ClC-c (fly homolog of CLCN5) causes Cubilin to accumulate in the ER and lose cortical localization in nephrocytes, with a strong decrease in albumin uptake. The authors speculate that ClC-c and V-ATPase together acidify the Golgi to allow proper glycosylation and surface trafficking of Cubilin or its binding partner Amnionless. ER retention of Cubilin was confirmed in ClC-5 knockout mice, supporting relevance to AMN-cubilin trafficking.","method":"Drosophila nephrocyte RNAi, immunolocalization of Cubilin and Amnionless, albumin uptake assay, ER marker colocalization, ClC-5 knockout mouse validation","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 — preprint; AMN is a secondary finding; mechanistic link between acidification and AMN glycosylation is speculative","pmids":[],"is_preprint":true}],"current_model":"AMN (amnionless) encodes a type I transmembrane protein that forms the obligate membrane-anchoring subunit of the cubam endocytic receptor complex: AMN binds the EGF domains of the peripheral membrane protein cubilin, mediates its ER export, promotes AMN-dependent N-linked glycosylation of cubilin required for surface targeting, delivers the complex to the apical membrane of polarized epithelia, and drives clathrin-mediated endocytosis of cubilin-bound ligands (including intrinsic factor-cobalamin) via FXNPXF motifs in its cytoplasmic tail that recruit the adaptor proteins Dab2 and ARH; loss of AMN function in intestine and kidney causes Imerslund-Gräsbeck syndrome, while in mouse visceral endoderm AMN is additionally required for middle primitive streak formation through a non-autonomous signaling mechanism."},"narrative":{"teleology":[{"year":1998,"claim":"Chimera experiments established that AMN functions cell-non-autonomously in the visceral endoderm to direct middle primitive streak formation, answering whether the gene acts within the embryo proper or in extra-embryonic tissue.","evidence":"ES cell↔blastocyst chimera analysis with amnionless-/- blastocysts and wild-type ES cells, histological and molecular phenotyping","pmids":["9851841"],"confidence":"Medium","gaps":["The downstream signaling pathway through which visceral endoderm AMN influences primitive streak assembly remains unidentified","Direct biochemical interaction of AMN with BMP ligands has not been demonstrated"]},{"year":2001,"claim":"Cloning of AMN revealed a type I transmembrane protein with a cysteine-rich ectodomain resembling BMP-binding modules, placing it at the intersection of transmembrane receptor biology and possible morphogen regulation.","evidence":"Gene cloning, transgene-induced insertional mutation, domain homology analysis, in situ hybridization in mouse embryos","pmids":["11279523"],"confidence":"Medium","gaps":["BMP-binding capacity was inferred from domain homology; direct ligand-binding experiments are lacking","Relationship between the putative BMP-binding domain and the cubilin-binding function was not addressed"]},{"year":2003,"claim":"Demonstration that AMN and cubilin form the cubam complex answered how the peripheral membrane protein cubilin reaches the cell surface and internalizes ligands, revealing AMN as both the membrane anchor and trafficking chaperone for cubilin.","evidence":"Co-purification by IF-cobalamin affinity chromatography and gel filtration; cotransfection of polarized epithelial cells; ligand endocytosis and lysosomal degradation assays","pmids":["14576052"],"confidence":"High","gaps":["Stoichiometry of the AMN–cubilin complex was not determined","Whether AMN itself contributes to ligand recognition was not resolved"]},{"year":2003,"claim":"Human genetic analysis showed that AMN mutations in exons 1–4 cause Imerslund–Gräsbeck syndrome without embryonic lethality, resolving the paradox between the severe mouse phenotype and human disease by demonstrating alternative downstream translation initiation.","evidence":"Mutation screening in IGS patients, translation initiation site mapping","pmids":["12590260"],"confidence":"Medium","gaps":["Precise functional domains of the truncated AMN isoform were not mapped","Whether the shorter AMN isoform has reduced affinity for cubilin was not tested"]},{"year":2004,"claim":"In vivo chimera studies in adult mice showed that AMN-null tissues fail to localize cubilin to the cell surface and exhibit selective proteinuria of cubilin ligands, establishing AMN as essential for cubilin surface targeting across multiple polarized epithelia.","evidence":"ES cell↔blastocyst chimeras, immunolocalization of cubilin in kidney and yolk sac, urinary protein analysis","pmids":["15342463"],"confidence":"High","gaps":["The intracellular fate of cubilin in AMN-null cells (ER vs. degradation) was not determined at this stage"]},{"year":2005,"claim":"Domain-mapping in polarized cells identified the cubilin EGF domains as the AMN-binding interface and showed AMN mediates ER export while cubilin contributes carbohydrate-dependent apical sorting signals, delineating the division of labor in cubam trafficking.","evidence":"Expression of cubilin and AMN domain fragments in MDCK cells, co-immunoprecipitation, tunicamycin treatment, apical vs. basolateral surface biotinylation","pmids":["15976000","15845892"],"confidence":"High","gaps":["Structural basis for the AMN–EGF domain interaction was not resolved","Identity of the ER quality-control checkpoint recognizing unpaired cubilin was not defined"]},{"year":2008,"claim":"Silencing experiments in kidney cells revealed that both AMN and megalin are independently required for cubilin intracellular stability, establishing a tripartite complex (cubilin–AMN–megalin) at the renal brush border.","evidence":"Ligand-affinity chromatography and co-IP of renal brush-border membranes, siRNA silencing of megalin and AMN, cubilin half-life measurement","pmids":["17990981"],"confidence":"High","gaps":["Whether cubilin degradation upon AMN loss proceeds through proteasomal or lysosomal pathways was not determined","Relative contributions of AMN versus megalin to cubilin stability were not quantitatively separated"]},{"year":2010,"claim":"Identification of two redundant FXNPXF motifs in the AMN cytoplasmic tail that recruit Dab2 and ARH explained the molecular basis for clathrin-mediated cubam endocytosis.","evidence":"Sequential mutagenesis of AMN FXNPXF motifs, yeast two-hybrid interaction with Dab2 and ARH, functional endocytosis assays","pmids":["20088845"],"confidence":"High","gaps":["Relative in vivo contribution of Dab2 versus ARH to cubam endocytosis in different tissues was not resolved","Phosphorylation or other post-translational regulation of the FXNPXF motifs was not assessed"]},{"year":2012,"claim":"Cross-species rescue of Drosophila nephrocyte protein uptake by human AMN demonstrated evolutionary conservation of AMN–cubilin co-receptor function, extending the paradigm beyond vertebrate polarized epithelia.","evidence":"Drosophila nephrocyte RNAi of dAMN, transgenic rescue with human AMN, protein uptake assays, electron microscopy","pmids":["23264686"],"confidence":"High","gaps":["Whether the AMN cytoplasmic endocytic signals are conserved in Drosophila was not tested","Ligand specificity of the fly cubam complex is poorly defined"]},{"year":2014,"claim":"Absence of megalin protein in human terminal ileum despite cubilin–AMN co-expression showed that cubam can function megalin-independently for intestinal vitamin B12 absorption, resolving tissue-specific receptor composition.","evidence":"Immunohistochemistry and quantitative RT-PCR for LRP2 in adult human terminal ileum","pmids":["25052491"],"confidence":"Medium","gaps":["Functional endocytosis data confirming megalin-independence in ileum were not provided","Whether an alternative endocytic co-receptor substitutes for megalin in ileum is unknown"]},{"year":2018,"claim":"Quantitative mass spectrometry and mutagenesis demonstrated that AMN-dependent N-linked glycosylation of cubilin—not merely physical association—is the critical step for ER-to-plasma-membrane trafficking, explaining how pathogenic missense mutations cause ER retention and disease.","evidence":"Quantitative membrane targeting assay, mass spectrometry of cubilin glycosylation sites, site-directed mutagenesis, patient-derived renal proximal tubular cell analysis","pmids":["29402915"],"confidence":"High","gaps":["How AMN promotes cubilin glycosylation mechanistically (chaperone vs. conformation-enabling) is not resolved","Structural model of the AMN–cubilin interface at the ER remains unavailable"]},{"year":null,"claim":"The structural basis for AMN–cubilin interaction, the mechanism by which AMN facilitates cubilin glycosylation, and the signaling pathway downstream of AMN in visceral endoderm remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No high-resolution structure of the AMN–cubilin complex exists","The embryonic signaling function of AMN has not been linked to a specific ligand or pathway","Tissue-specific regulatory mechanisms controlling AMN expression are largely undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0038024","term_label":"cargo receptor activity","supporting_discovery_ids":[2,8,10]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,6,8]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,4,5,6,12]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[6,12]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,8,10]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[6,12]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[2,3,9]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,1]}],"complexes":["cubam (cubilin–AMN)"],"partners":["CUBN","LRP2","DAB2","LDLRAP1"],"other_free_text":[]},"mechanistic_narrative":"AMN (amnionless) is the obligate transmembrane anchor of the cubam endocytic receptor complex, essential for vitamin B12 absorption in the intestine and protein reabsorption in the kidney. AMN binds the EGF domains of cubilin, promotes its N-linked glycosylation, mediates ER export, and delivers the complex to the apical plasma membrane of polarized epithelia; without AMN, cubilin is retained in the ER and fails to reach the cell surface [PMID:14576052, PMID:29402915, PMID:15845892]. Two cytoplasmic FXNPXF motifs in AMN recruit the clathrin-associated adaptors Dab2 and ARH to drive endocytosis of cubilin-bound ligands including intrinsic factor–cobalamin [PMID:20088845]. Loss-of-function mutations in AMN cause Imerslund–Gräsbeck syndrome (selective intestinal vitamin B12 malabsorption with proteinuria), and in mouse visceral endoderm AMN is additionally required non-autonomously for middle primitive streak formation [PMID:12590260, PMID:9851841]."},"prefetch_data":{"uniprot":{"accession":"Q9BXJ7","full_name":"Protein amnionless","aliases":[],"length_aa":453,"mass_kda":47.8,"function":"Membrane-bound component of the endocytic receptor formed by AMN and CUBN (PubMed:14576052, PubMed:29402915, PubMed:30523278). Required for normal CUBN glycosylation and trafficking to the cell surface (PubMed:14576052, PubMed:29402915). The complex formed by AMN and CUBN is required for efficient absorption of vitamin B12 (PubMed:12590260, PubMed:14576052, PubMed:26040326). Required for normal CUBN-mediated protein transport in the kidney (Probable)","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/Q9BXJ7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AMN","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/AMN","total_profiled":1310},"omim":[{"mim_id":"618882","title":"IMERSLUND-GRASBECK SYNDROME 2; IGS2","url":"https://www.omim.org/entry/618882"},{"mim_id":"614362","title":"ACYL-CoA SYNTHETASE, BUBBLEGUM FAMILY, MEMBER 1; ACSBG1","url":"https://www.omim.org/entry/614362"},{"mim_id":"614071","title":"MYOCARDIAL ZONULA ADHERENS PROTEIN; MYZAP","url":"https://www.omim.org/entry/614071"},{"mim_id":"609342","title":"COBALAMIN-BINDING INTRINSIC FACTOR; CBLIF","url":"https://www.omim.org/entry/609342"},{"mim_id":"605799","title":"AMNION-ASSOCIATED TRANSMEMBRANE PROTEIN; AMN","url":"https://www.omim.org/entry/605799"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"intestine","ntpm":86.9},{"tissue":"kidney","ntpm":52.4},{"tissue":"liver","ntpm":55.2}],"url":"https://www.proteinatlas.org/search/AMN"},"hgnc":{"alias_symbol":["amnionless"],"prev_symbol":[]},"alphafold":{"accession":"Q9BXJ7","domains":[{"cath_id":"-","chopping":"25-104","consensus_level":"medium","plddt":89.7134,"start":25,"end":104},{"cath_id":"-","chopping":"119-226","consensus_level":"medium","plddt":92.8303,"start":119,"end":226},{"cath_id":"3.30.70,3.30.70","chopping":"235-350","consensus_level":"high","plddt":91.5153,"start":235,"end":350}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BXJ7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BXJ7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BXJ7-F1-predicted_aligned_error_v6.png","plddt_mean":80.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AMN","jax_strain_url":"https://www.jax.org/strain/search?query=AMN"},"sequence":{"accession":"Q9BXJ7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BXJ7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BXJ7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BXJ7"}},"corpus_meta":[{"pmid":"14576052","id":"PMC_14576052","title":"The functional cobalamin (vitamin B12)-intrinsic factor receptor is a novel complex of cubilin and amnionless.","date":"2003","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/14576052","citation_count":219,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"12590260","id":"PMC_12590260","title":"Amnionless, essential for mouse gastrulation, is mutated in recessive hereditary megaloblastic anemia.","date":"2003","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/12590260","citation_count":108,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11279523","id":"PMC_11279523","title":"The amnionless gene, essential for mouse gastrulation, encodes a visceral-endoderm-specific protein with an extracellular cysteine-rich domain.","date":"2001","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/11279523","citation_count":107,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"15845892","id":"PMC_15845892","title":"Amnionless function is required for cubilin brush-border expression and intrinsic factor-cobalamin (vitamin B12) absorption in vivo.","date":"2005","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/15845892","citation_count":94,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"23264686","id":"PMC_23264686","title":"Cubilin and amnionless mediate protein reabsorption in Drosophila nephrocytes.","date":"2012","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/23264686","citation_count":88,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"15976000","id":"PMC_15976000","title":"Contribution of cubilin and amnionless to processing and membrane targeting of cubilin-amnionless complex.","date":"2005","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/15976000","citation_count":73,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"15342463","id":"PMC_15342463","title":"Mouse amnionless, which is required for primitive streak assembly, mediates cell-surface localization and endocytic function of cubilin on visceral endoderm and kidney proximal tubules.","date":"2004","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/15342463","citation_count":69,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"10516293","id":"PMC_10516293","title":"Developmental expression of an amn(+) transgene rescues the mutant memory defect of amnesiac adults.","date":"1999","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/10516293","citation_count":68,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"17990981","id":"PMC_17990981","title":"Interactions of cubilin with megalin and the product of the amnionless gene (AMN): effect on its stability.","date":"2008","source":"The Biochemical 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\"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (biochemical co-purification, colocalization, functional cell transfection assays), strong mechanistic conclusion, highly cited\",\n      \"pmids\": [\"14576052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"AMN is a type I transmembrane protein with an extracellular cysteine-rich domain; homozygous mutations in exons 1-4 cause selective vitamin B12 malabsorption (Imerslund-Gräsbeck syndrome), and when the 5' end is truncated by mutations, translation initiates from alternative downstream start codons.\",\n      \"method\": \"Genetic mapping, mutation analysis by sequencing, identification of alternative translation initiation in patient cells\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic and molecular characterization replicated across multiple families, foundational domain architecture paper with >100 citations\",\n      \"pmids\": [\"12590260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"AMN encodes a novel type I transmembrane protein expressed exclusively in extra-embryonic visceral endoderm during gastrulation; loss of AMN function in the visceral endoderm (established by chimera analysis) impairs middle primitive streak assembly, suggesting AMN modulates a BMP signaling pathway in the visceral endoderm.\",\n      \"method\": \"Gene cloning, expression analysis, transgene-insertion mouse mutant phenotypic characterization, chimera analysis\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — original cloning paper with chimera functional analysis, >100 citations, replicated in multiple subsequent studies\",\n      \"pmids\": [\"11279523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"AMN function is required in vivo for cubilin processing and apical (brush border) membrane targeting; loss of AMN expression (due to in-frame deletion or initiation codon mutation in canine AMN) blocks cubilin targeting to the apical membrane, abrogating IF-cobalamin endocytosis.\",\n      \"method\": \"In vivo canine model with AMN mutations, immunohistochemistry, cell-transfection validation system\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic model validated orthogonally in cell transfection system, mechanistically links AMN to cubilin membrane targeting\",\n      \"pmids\": [\"15845892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"AMN is required for cubilin cell-surface localization and endocytic function in kidney proximal tubules and visceral endoderm in vivo; Amn-/- ES cell chimeras show selective proteinuria of cubilin ligands and mislocalization of cubilin away from the cell surface in Amn-/- tissues.\",\n      \"method\": \"Amn-/- ES cell↔wildtype blastocyst chimeras, immunolocalization, proteinuria analysis\",\n      \"journal\": \"Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — rigorous in vivo chimera genetic approach with defined cellular phenotype and protein localization data\",\n      \"pmids\": [\"15342463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"AMN interacts with the EGF domains of cubilin (not the N-terminal domain), provides membrane anchorage, and drives export of the cubilin-AMN complex from the endoplasmic reticulum; apical sorting of the complex depends on cubilin's extracellular carbohydrate moieties.\",\n      \"method\": \"Expression of AMN and cubilin truncation/deletion mutants in polarized MDCK cells, co-precipitation, confocal colocalization, tunicamycin glycosylation inhibition\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — systematic mutagenesis combined with biochemical and imaging approaches in a polarized epithelial model\",\n      \"pmids\": [\"15976000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The cytosolic domain of AMN contains two FXNPXF motifs that are functionally redundant; both signals mediate endocytosis of the cubam receptor complex through direct interaction with the clathrin-associated sorting proteins Dab2 and ARH.\",\n      \"method\": \"Sequential mutagenesis of AMN FXNPXF signals, expression of AMN mutant panel in cells, yeast two-hybrid analysis with Dab2 and ARH\",\n      \"journal\": \"Traffic\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — systematic mutagenesis combined with yeast two-hybrid and functional endocytosis assays, defines the molecular endocytic mechanism\",\n      \"pmids\": [\"20088845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Cubilin interacts with both AMN and megalin in renal brush border membranes; both interactions are essential for cubilin intracellular stability, as silencing of either megalin or AMN by gene-silencing in opossum kidney cells reduces cubilin levels by 85-90% and decreases its half-life ~2-fold.\",\n      \"method\": \"In vitro binding assays with cubilin fragments and megalin, co-immunoprecipitation from rat kidney brush border membranes, IF-cobalamin affinity chromatography, siRNA knockdown in opossum kidney cells with turnover studies\",\n      \"journal\": \"Biochemical Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal biochemical methods (pulldown, Co-IP, affinity chromatography, KD with turnover) converging on the same conclusion\",\n      \"pmids\": [\"17990981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"AMN-mediated N-linked glycosylation of cubilin (at least 4 specific residues) is necessary for surface targeting of the cubilin-AMN complex; interaction between cubilin and AMN alone is not sufficient — glycosylation of cubilin by AMN is required for plasma membrane expression.\",\n      \"method\": \"Quantitative surface-expression assay in renal and intestinal cell lines, quantitative mass spectrometry to identify glycosylation sites, mutagenesis of glycosylation residues, patient proximal tubular cell analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis combined with MS glycosylation site mapping and patient cell validation, defines a novel AMN function in glycosylation-dependent trafficking\",\n      \"pmids\": [\"29402915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Drosophila orthologs of cubilin and AMN (dCubilin and dAMN) function as co-receptors for protein reabsorption specifically in nephrocytes; human AMN can rescue defective protein uptake caused by dAMN knockdown in fly nephrocytes, demonstrating evolutionary conservation of the cubilin/AMN co-receptor function.\",\n      \"method\": \"Drosophila genetic screen, RNAi knockdown, targeted expression of human AMN in nephrocytes, functional protein uptake assay\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cross-species rescue experiment demonstrates functional conservation; RNAi with defined phenotypic readout\",\n      \"pmids\": [\"23264686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The amn gene product is required specifically in extraembryonic visceral endoderm (not in the embryo proper) for the generation of middle primitive streak derivatives, as demonstrated by wildtype ES cell↔amnionless-/- blastocyst chimera analysis.\",\n      \"method\": \"Insertional mouse mutant characterization, wildtype ES cell↔amn-/- blastocyst chimera analysis, histological and molecular analysis\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — chimera genetic epistasis cleanly localizes AMN function to visceral endoderm with defined mesodermal phenotypic consequence\",\n      \"pmids\": [\"9851841\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In adult human terminal ileum, cubilin and AMN colocalize in epithelial cells and function independently of megalin (LRP2), which is absent at the protein level despite its presence in kidney; this indicates tissue-specific variation in the cubilin-AMN endocytic mechanism.\",\n      \"method\": \"Immunohistochemistry, RT-PCR for LRP2/cubilin/AMN mRNA, protein expression analysis in adult human terminal ileum sections\",\n      \"journal\": \"Physiological reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single study with protein/mRNA expression data in human tissue; no functional assay, but provides direct localization evidence with mechanistic implication\",\n      \"pmids\": [\"25052491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In Drosophila nephrocytes, store-operated calcium entry (SOCE) via Stim/Orai mediates the proper localization of Amnionless (dAMN) at the cell cortex; SOCE inhibition or Stim/Orai knockdown reduces dAMN abundance and disrupts albumin endocytosis.\",\n      \"method\": \"GCaMP6 live calcium imaging in nephrocytes, EGTA/2-APB pharmacology, RNAi knockdown of Stim and Orai, albumin uptake assay, immunofluorescence for dAMN localization\",\n      \"journal\": \"Journal of insect physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional calcium imaging combined with genetic knockdown and protein localization; ortholog study in Drosophila with relevance to mammalian AMN\",\n      \"pmids\": [\"36341969\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AMN (amnionless) is a type I transmembrane protein that forms the cubam receptor complex with cubilin by binding to cubilin's EGF domains, driving ER export, mediating N-linked glycosylation of cubilin required for surface targeting, anchoring the complex to the apical plasma membrane, and directing clathrin-mediated endocytosis of cubilin with its ligands (including intrinsic factor-cobalamin) via cytosolic FXNPXF motifs that recruit the sorting adaptors Dab2 and ARH; loss of AMN function abolishes cubilin surface localization and endocytosis in intestinal and renal epithelia, causing Imerslund-Gräsbeck syndrome, while in early mouse development AMN acts in visceral endoderm to direct middle primitive streak formation.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll systematically classify each paper before extracting discoveries.\n\n**Classification:**\n\n- Papers about AMN (amnionless gene/protein, cubilin receptor complex, Imerslund-Gräsbeck syndrome): KEEP\n- Papers about AMN as \"adrenomyeloneuropathy\" (phenotype of ABCD1/X-ALD): EXCLUDE (alias collision)\n- Papers about AMN-107 (nilotinib drug): EXCLUDE (alias collision)\n- Papers about \"AMN\" as action-mode network (neuroscience): EXCLUDE (alias collision)\n- Papers about \"AMN\" as autobiographical memory network: EXCLUDE (alias collision)\n- Papers about AMN nucleosidase in E. coli: EXCLUDE (symbol collision, microbial)\n- Papers about AMN in Corynebacterium glutamicum: EXCLUDE (symbol collision, microbial)\n- Papers about AMN kagome lattice materials: EXCLUDE (symbol collision)\n- Papers about amnesiac (amn) in Drosophila memory: EXCLUDE (different gene - Drosophila amnesiac encodes PACAP-like neuropeptide, not AMN ortholog)\n- Drosophila cubilin/amnionless paper (PMID:23264686): KEEP (ortholog)\n- BioPlex/interactome papers: EXCLUDE (not mechanistic about AMN specifically)\n- General cDNA/genome papers: EXCLUDE\n\n**KEPT papers:** 14576052, 12590260, 11279523, 15845892, 23264686, 15976000, 15342463, 17990981, 20088845, 29057905, 29402915, 8660894 (EXCLUDE - about TRAF protein candidate, not AMN itself), 25052491, 21750092, 18181028, 21974783, 36341969, bio_10.1101_2025.03.05.641445\n\nNote: PMID 8660894 describes a TRAF-related protein as a \"candidate\" for the amnionless gene but identifies TRAFamn (which is TRAF3), not the actual AMN protein - EXCLUDE.\nPMID 9851841: describes amn mutant phenotype in visceral endoderm - KEEP (functional role).\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"AMN encodes a novel type I transmembrane protein expressed exclusively in the extra-embryonic visceral endoderm during mouse gastrulation. The extracellular region contains a cysteine-rich domain with similarity to BMP-binding domains in chordin and Short gastrulation, suggesting AMN may modulate a BMP signaling pathway in the visceral endoderm to direct trunk mesoderm formation from the middle primitive streak.\",\n      \"method\": \"Gene cloning, transgene-induced insertional mutation, in situ hybridization, domain analysis, mouse embryo phenotyping\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct loss-of-function phenotype with localization, but BMP-binding is inferred from domain homology, not directly demonstrated\",\n      \"pmids\": [\"11279523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The amn gene product is required in extraembryonic tissues (visceral endoderm) for the generation of middle primitive streak derivatives. Chimera experiments using wild-type ES cells in amnionless-/- blastocysts demonstrated that the amnionless function in visceral endoderm is cell-non-autonomous with respect to the embryo proper, establishing a signaling role for AMN in the visceral endoderm during gastrulation.\",\n      \"method\": \"ES cell↔blastocyst chimera analysis, histological and molecular phenotyping of amnionless mutant embryos\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis via chimera experiment with defined tissue-specific requirement\",\n      \"pmids\": [\"9851841\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Cubilin and AMN form a tight heteromeric complex (cubam). AMN binds to the amino-terminal third of cubilin, directs cubilin trafficking from early biosynthetic compartments to the cell surface and endosomes, and enables intrinsic factor-cobalamin endocytosis and lysosomal degradation. Neither protein alone confers ligand endocytosis; cotransfection of both is required for functional receptor activity.\",\n      \"method\": \"Co-purification by IF-cobalamin affinity chromatography and non-denaturing gel filtration; transfection of polarized epithelial cells; colocalization by immunofluorescence; ligand endocytosis and lysosomal degradation assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — biochemical co-purification plus functional reconstitution in transfected cells with multiple orthogonal readouts\",\n      \"pmids\": [\"14576052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Homozygous mutations affecting exons 1–4 of human AMN cause selective intestinal malabsorption of vitamin B12 (Imerslund-Gräsbeck syndrome) without affecting embryonic development, indicating the 5′ end of AMN is dispensable for gastrulation but necessary for cobalamin absorption. When the 5′ end is truncated, translation initiates from alternative downstream start codons.\",\n      \"method\": \"Human genetic mutation analysis, translation initiation site mapping\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — human loss-of-function genetics with defined molecular consequence (alternative translation initiation)\",\n      \"pmids\": [\"12590260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"AMN is expressed in kidney proximal tubules and intestinal epithelium in addition to visceral endoderm. In Amn−/− ES cell↔wild-type blastocyst chimeras, Cubn is not properly localized to the cell surface in Amn−/− tissues, adult chimeras exhibit selective proteinuria of Cubn ligands, and Amn−/− embryos show failure of epiblast growth linked to middle primitive streak assembly. This establishes AMN as an essential in vivo component of the Cubn receptor complex mediating endocytosis/transcytosis in polarized epithelia.\",\n      \"method\": \"ES cell↔blastocyst chimera generation, immunolocalization of Cubn in embryo and adult mouse tissues, urinary protein analysis\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic loss-of-function with direct protein localization and functional (proteinuria) readout, replicated across tissue types\",\n      \"pmids\": [\"15342463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"In vivo canine models of I-GS with AMN mutations (in-frame deletion in exon 10 or substitution in the translation initiation codon) demonstrate that loss of AMN expression blocks cubilin processing and targeting to the apical brush-border membrane, abolishing functional receptor activity. These features are recapitulated in a heterologous cell-transfection system, validating AMN's essential role in cubilin apical membrane targeting.\",\n      \"method\": \"Canine genetic model, in vivo tissue studies, heterologous cell transfection, immunohistochemistry\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo animal model with defined mutations, direct protein localization, replicated in cell-based system\",\n      \"pmids\": [\"15845892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"AMN interacts with the EGF domains of cubilin (not the N-terminal domain) and is responsible for membrane attachment and ER export of the cubilin-AMN complex. Cubilin itself mediates apical sorting in a carbohydrate-dependent manner (sensitive to tunicamycin). Truncated cubilin constructs lacking the N-terminal domain reach the apical membrane only when co-expressed with AMN.\",\n      \"method\": \"Expression of AMN and cubilin fragments in polarized MDCK cells, co-immunoprecipitation, colocalization, tunicamycin treatment\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic domain-mapping with co-IP and functional trafficking readout in polarized cells\",\n      \"pmids\": [\"15976000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In renal brush-border membranes, cubilin exists in a functional complex with both AMN and megalin. Three distinct regions of cubilin (N-terminus, CUB domains 12–17, CUB domains 22–27) show Ca2+-dependent binding to megalin in vitro. Silencing megalin or AMN in opossum kidney cells reduces cubilin levels by 85–90% and decreases cubilin half-life 2-fold, indicating that interactions with both megalin and AMN are required for cubilin intracellular stability.\",\n      \"method\": \"Ligand-affinity chromatography, immunoprecipitation of renal brush-border membranes, in vitro Ca2+-dependent binding assays, gene silencing (siRNA), turnover/half-life studies, immunohistochemistry\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including in vitro binding, co-IP of native membranes, and functional gene silencing with quantitative protein stability readout\",\n      \"pmids\": [\"17990981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The cytosolic domain of AMN contains two FXNPXF motifs that are functionally redundant and mediate cubam endocytosis by interacting with the clathrin-associated sorting proteins Dab2 and ARH. Sequential mutagenesis of each motif combined with yeast two-hybrid analysis demonstrated that either signal alone is sufficient to drive endocytosis through Dab2 or ARH.\",\n      \"method\": \"Sequential mutagenesis of AMN cytoplasmic FXNPXF motifs, expression of AMN mutant panel, yeast two-hybrid interaction assays, functional endocytosis assays\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis combined with yeast two-hybrid and functional endocytosis assay, defining molecular endocytic mechanism\",\n      \"pmids\": [\"20088845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Compound heterozygous AMN mutations (premature termination codons in exons 6 and 7) dramatically decrease cubilin receptor activity at the luminal surface of intestinal cells in humans, despite normal cubilin affinity for intrinsic factor. This demonstrates that AMN is essential for correct luminal (apical) expression of cubilin in vivo.\",\n      \"method\": \"Human genetic analysis, measurement of cubilin receptor activity in urine, intrinsic factor binding affinity assay\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — human loss-of-function with direct functional receptor activity measurement, but single case study\",\n      \"pmids\": [\"21750092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Drosophila orthologs of cubilin (dCubilin) and amnionless (dAMN) are specifically expressed in nephrocytes where they function as co-receptors for protein uptake. Human AMN expressed in Drosophila nephrocytes rescues the protein-uptake defect caused by dAMN knockdown, demonstrating evolutionary conservation of cubilin/AMN co-receptor function. Cubilin/AMN-mediated protein reabsorption is required for maintenance of nephrocyte ultrastructure and fly survival under toxic stress.\",\n      \"method\": \"Genetic screen, targeted RNAi knockdown in Drosophila nephrocytes, transgenic rescue with human AMN, protein uptake assays, electron microscopy of ultrastructure\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cross-species functional rescue experiment with multiple readouts (protein uptake, ultrastructure, survival), confirming conserved mechanism\",\n      \"pmids\": [\"23264686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In adult human terminal ileum, cubilin and AMN co-localize to epithelial cells but megalin protein is absent (with extremely low LRP2 mRNA). This demonstrates that cubilin and AMN can function independently of megalin for vitamin B12 absorption in the intestine, establishing tissue-specific and ligand-specific dependence on megalin.\",\n      \"method\": \"Immunohistochemistry, quantitative RT-PCR for LRP2 mRNA in adult human terminal ileum tissue\",\n      \"journal\": \"Physiological reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — direct protein localization in human tissue, but mechanistic inference rather than functional experiment\",\n      \"pmids\": [\"25052491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"AMN-mediated glycosylation of cubilin is necessary (not merely AMN-cubilin interaction) for plasma membrane expression of the complex. N-linked glycosylation of at least 4 cubilin residues is required for surface targeting. Pathogenic AMN and CUBN missense mutations cause ER retention and completely block amnionless-dependent plasma membrane expression of cubilin, as confirmed in renal proximal tubular cells from an IGS patient.\",\n      \"method\": \"Quantitative membrane targeting assay in cultured renal and intestinal cells, quantitative mass spectrometry, site-directed mutagenesis of N-glycosylation sites, patient-derived proximal tubular cell analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis plus mass spectrometry plus patient cell validation, with quantitative functional readout\",\n      \"pmids\": [\"29402915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In Drosophila nephrocytes, store-operated calcium entry (SOCE) mediated by Stim/Orai regulates the abundance and localization of dAMN (the amnionless ortholog). RNAi knockdown of Stim or Orai reduced dAMN abundance, impaired albumin binding and endocytosis, and disrupted slit diaphragm organization. Calcium chelation (EGTA) reduced albumin binding following pre-treatment, linking SOCE to AMN-dependent endocytic function.\",\n      \"method\": \"GCaMP6 calcium imaging in vivo and in vitro, RNAi knockdown of SOCE genes (Stim, Orai), pharmacological inhibition (EGTA, 2-APB), albumin uptake assay, immunolocalization of dAMN\",\n      \"journal\": \"Journal of insect physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic knockdown with calcium imaging and functional endocytosis readout in Drosophila ortholog model\",\n      \"pmids\": [\"36341969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In a Drosophila model of Dent's disease, depletion of ClC-c (fly homolog of CLCN5) causes Cubilin to accumulate in the ER and lose cortical localization in nephrocytes, with a strong decrease in albumin uptake. The authors speculate that ClC-c and V-ATPase together acidify the Golgi to allow proper glycosylation and surface trafficking of Cubilin or its binding partner Amnionless. ER retention of Cubilin was confirmed in ClC-5 knockout mice, supporting relevance to AMN-cubilin trafficking.\",\n      \"method\": \"Drosophila nephrocyte RNAi, immunolocalization of Cubilin and Amnionless, albumin uptake assay, ER marker colocalization, ClC-5 knockout mouse validation\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — preprint; AMN is a secondary finding; mechanistic link between acidification and AMN glycosylation is speculative\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"AMN (amnionless) encodes a type I transmembrane protein that forms the obligate membrane-anchoring subunit of the cubam endocytic receptor complex: AMN binds the EGF domains of the peripheral membrane protein cubilin, mediates its ER export, promotes AMN-dependent N-linked glycosylation of cubilin required for surface targeting, delivers the complex to the apical membrane of polarized epithelia, and drives clathrin-mediated endocytosis of cubilin-bound ligands (including intrinsic factor-cobalamin) via FXNPXF motifs in its cytoplasmic tail that recruit the adaptor proteins Dab2 and ARH; loss of AMN function in intestine and kidney causes Imerslund-Gräsbeck syndrome, while in mouse visceral endoderm AMN is additionally required for middle primitive streak formation through a non-autonomous signaling mechanism.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"AMN (amnionless) is a type I transmembrane protein that functions as the essential membrane-anchoring and trafficking subunit of the cubam receptor complex, which mediates endocytic uptake of intrinsic factor–cobalamin and other ligands in intestinal and renal epithelia. AMN binds cubilin's EGF domains, drives ER export of the complex, promotes N-linked glycosylation of cubilin required for apical surface targeting, and anchors cubilin at the plasma membrane; its cytosolic FXNPXF motifs recruit the clathrin-associated sorting adaptors Dab2 and ARH to mediate endocytosis [PMID:14576052, PMID:15976000, PMID:20088845, PMID:29402915]. Loss-of-function mutations in AMN abolish cubilin surface localization and cause Imerslund–Gräsbeck syndrome (selective vitamin B12 malabsorption with proteinuria) [PMID:12590260, PMID:15845892, PMID:15342463]. In early mouse development, AMN acts in the visceral endoderm to direct middle primitive streak formation, indicating a distinct developmental signaling role [PMID:9851841, PMID:11279523].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing that AMN functions non-cell-autonomously in extraembryonic visceral endoderm to specify middle primitive streak derivatives resolved the tissue of action for the amnionless developmental phenotype.\",\n      \"evidence\": \"Wildtype ES cell↔amn−/− blastocyst chimera analysis in mouse with histological and molecular assessment\",\n      \"pmids\": [\"9851841\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The signaling pathway downstream of AMN in visceral endoderm was not identified\",\n        \"Whether AMN acts as a receptor or a co-receptor for a developmental ligand was unclear\"\n      ]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Cloning of AMN revealed it encodes a novel type I transmembrane protein with a cysteine-rich extracellular domain expressed in visceral endoderm, and suggested it modulates BMP signaling during gastrulation.\",\n      \"evidence\": \"Gene cloning, expression analysis, and phenotypic characterization of transgene-insertion mouse mutant with chimera analysis\",\n      \"pmids\": [\"11279523\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct interaction with BMP pathway components was not demonstrated\",\n        \"The relationship between AMN's developmental and epithelial functions was unexplained\"\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Discovery that AMN and cubilin form the cubam complex and that AMN directs cubilin from biosynthetic compartments to the cell surface and endosomes established AMN as the trafficking subunit of the receptor, while concurrent identification of AMN mutations in Imerslund–Gräsbeck syndrome families linked this function to human disease.\",\n      \"evidence\": \"Co-purification by IF-cobalamin affinity chromatography, colocalization, functional endocytosis assays in polarized epithelial cells; patient mutation analysis and alternative translation initiation characterization\",\n      \"pmids\": [\"14576052\", \"12590260\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The specific cubilin domains contacted by AMN were not mapped\",\n        \"The endocytic signals within AMN were not yet identified\"\n      ]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"In vivo chimera studies demonstrated that AMN is required for cubilin cell-surface localization and endocytic function in kidney proximal tubules and visceral endoderm, with Amn−/− cells showing selective proteinuria of cubilin ligands.\",\n      \"evidence\": \"Amn−/− ES cell↔wildtype blastocyst chimeras with immunolocalization and proteinuria analysis\",\n      \"pmids\": [\"15342463\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether AMN loss affects cubilin protein stability versus trafficking alone was not resolved\"\n      ]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Mapping the AMN–cubilin interaction to cubilin's EGF domains (not the N-terminal domain) and showing that AMN drives ER export of the complex defined the domain architecture and the rate-limiting trafficking step, while canine AMN mutations confirmed this mechanism in vivo.\",\n      \"evidence\": \"Truncation/deletion mutagenesis in polarized MDCK cells with co-precipitation and confocal imaging; canine AMN mutant model with immunohistochemistry\",\n      \"pmids\": [\"15976000\", \"15845892\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The ER export signal or chaperone mechanism used by AMN was not identified\",\n        \"Structural basis of AMN–cubilin EGF domain interaction was unknown\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrating that both AMN and megalin are required for cubilin intracellular stability in kidney cells, with silencing of either reducing cubilin levels by ~90%, revealed that cubam function depends on a tripartite complex.\",\n      \"evidence\": \"In vitro binding assays, co-immunoprecipitation from rat kidney brush border membranes, siRNA knockdown with protein turnover studies in opossum kidney cells\",\n      \"pmids\": [\"17990981\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether megalin and AMN interact directly or only via cubilin was not resolved\",\n        \"The degradation pathway for cubilin in the absence of AMN was not characterized\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identification of two functionally redundant FXNPXF motifs in AMN's cytosolic tail that directly recruit Dab2 and ARH defined the molecular mechanism by which cubam undergoes clathrin-mediated endocytosis.\",\n      \"evidence\": \"Sequential mutagenesis of AMN FXNPXF signals, functional endocytosis assays, yeast two-hybrid with Dab2 and ARH\",\n      \"pmids\": [\"20088845\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Relative contributions of Dab2 versus ARH in different tissues were not determined\",\n        \"Whether additional cytosolic signals contribute to endocytic sorting was unknown\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrating that Drosophila AMN/cubilin orthologs function as co-receptors for protein reabsorption in nephrocytes, with human AMN rescuing dAMN loss, established deep evolutionary conservation of the cubam endocytic mechanism.\",\n      \"evidence\": \"Drosophila RNAi knockdown, cross-species rescue by human AMN expression, functional protein uptake assay in nephrocytes\",\n      \"pmids\": [\"23264686\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether Drosophila cubam handles the same ligand spectrum as mammalian cubam was not tested\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Observation that cubilin and AMN colocalize in human terminal ileum epithelium where megalin protein is absent indicated that cubam functions independently of megalin in the intestine, revealing tissue-specific variation in the endocytic mechanism.\",\n      \"evidence\": \"Immunohistochemistry and RT-PCR for LRP2/cubilin/AMN in adult human ileum sections\",\n      \"pmids\": [\"25052491\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No functional endocytosis assay was performed in ileal tissue\",\n        \"The alternative endocytic co-receptor (if any) replacing megalin in ileum was not identified\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showing that AMN-mediated N-linked glycosylation of cubilin at specific residues is required for surface targeting — beyond mere physical interaction — added a post-translational quality-control step to the cubam trafficking model.\",\n      \"evidence\": \"Quantitative surface expression assays, mass spectrometry glycosylation site mapping, mutagenesis of glycosylation residues, patient proximal tubular cell analysis\",\n      \"pmids\": [\"29402915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether AMN directly promotes glycosylation or indirectly enables it via ER retention was not distinguished\",\n        \"The glycosyltransferases acting on cubilin in the AMN-dependent step were not identified\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linking store-operated calcium entry via Stim/Orai to AMN cortical localization in Drosophila nephrocytes introduced calcium signaling as an upstream regulator of cubam surface expression.\",\n      \"evidence\": \"GCaMP6 calcium imaging, pharmacological inhibition, RNAi of Stim/Orai, albumin uptake and immunofluorescence in Drosophila nephrocytes\",\n      \"pmids\": [\"36341969\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The mechanism by which SOCE regulates AMN localization (direct or indirect) is unknown\",\n        \"Relevance to mammalian AMN trafficking has not been tested\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"No structural model of the AMN–cubilin interface exists, and the mechanism by which AMN promotes cubilin glycosylation remains unresolved; the relationship between AMN's developmental signaling role in visceral endoderm and its epithelial receptor-trafficking function is also unexplained.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No crystal or cryo-EM structure of the AMN–cubilin complex\",\n        \"Mechanism connecting AMN to BMP signaling in visceral endoderm is undefined\",\n        \"Whether AMN has ligand-binding activity independent of cubilin is unknown\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\n        \"term_id\": \"GO:0060090\",\n        \"supporting_discovery_ids\": [0, 5, 6]\n      },\n      {\n        \"term_id\": \"GO:0038024\",\n        \"supporting_discovery_ids\": [0, 6, 9]\n      }\n    ],\n    \"localization\": [\n      {\n        \"term_id\": \"GO:0005886\",\n        \"supporting_discovery_ids\": [0, 3, 4, 5, 8]\n      },\n      {\n        \"term_id\": \"GO:0005783\",\n        \"supporting_discovery_ids\": [5, 8]\n      },\n      {\n        \"term_id\": \"GO:0005768\",\n        \"supporting_discovery_ids\": [0]\n      }\n    ],\n    \"pathway\": [\n      {\n        \"term_id\": \"R-HSA-5653656\",\n        \"supporting_discovery_ids\": [0, 6, 9]\n      },\n      {\n        \"term_id\": \"R-HSA-392499\",\n        \"supporting_discovery_ids\": [8]\n      },\n      {\n        \"term_id\": \"R-HSA-382551\",\n        \"supporting_discovery_ids\": [0, 1, 3]\n      }\n    ],\n    \"complexes\": [\n      \"cubam (cubilin-AMN complex)\"\n    ],\n    \"partners\": [\n      \"CUBN\",\n      \"LRP2\",\n      \"DAB2\",\n      \"LDLRAP1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"AMN (amnionless) is the obligate transmembrane anchor of the cubam endocytic receptor complex, essential for vitamin B12 absorption in the intestine and protein reabsorption in the kidney. AMN binds the EGF domains of cubilin, promotes its N-linked glycosylation, mediates ER export, and delivers the complex to the apical plasma membrane of polarized epithelia; without AMN, cubilin is retained in the ER and fails to reach the cell surface [PMID:14576052, PMID:29402915, PMID:15845892]. Two cytoplasmic FXNPXF motifs in AMN recruit the clathrin-associated adaptors Dab2 and ARH to drive endocytosis of cubilin-bound ligands including intrinsic factor–cobalamin [PMID:20088845]. Loss-of-function mutations in AMN cause Imerslund–Gräsbeck syndrome (selective intestinal vitamin B12 malabsorption with proteinuria), and in mouse visceral endoderm AMN is additionally required non-autonomously for middle primitive streak formation [PMID:12590260, PMID:9851841].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Chimera experiments established that AMN functions cell-non-autonomously in the visceral endoderm to direct middle primitive streak formation, answering whether the gene acts within the embryo proper or in extra-embryonic tissue.\",\n      \"evidence\": \"ES cell↔blastocyst chimera analysis with amnionless-/- blastocysts and wild-type ES cells, histological and molecular phenotyping\",\n      \"pmids\": [\"9851841\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The downstream signaling pathway through which visceral endoderm AMN influences primitive streak assembly remains unidentified\",\n        \"Direct biochemical interaction of AMN with BMP ligands has not been demonstrated\"\n      ]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Cloning of AMN revealed a type I transmembrane protein with a cysteine-rich ectodomain resembling BMP-binding modules, placing it at the intersection of transmembrane receptor biology and possible morphogen regulation.\",\n      \"evidence\": \"Gene cloning, transgene-induced insertional mutation, domain homology analysis, in situ hybridization in mouse embryos\",\n      \"pmids\": [\"11279523\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"BMP-binding capacity was inferred from domain homology; direct ligand-binding experiments are lacking\",\n        \"Relationship between the putative BMP-binding domain and the cubilin-binding function was not addressed\"\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstration that AMN and cubilin form the cubam complex answered how the peripheral membrane protein cubilin reaches the cell surface and internalizes ligands, revealing AMN as both the membrane anchor and trafficking chaperone for cubilin.\",\n      \"evidence\": \"Co-purification by IF-cobalamin affinity chromatography and gel filtration; cotransfection of polarized epithelial cells; ligand endocytosis and lysosomal degradation assays\",\n      \"pmids\": [\"14576052\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Stoichiometry of the AMN–cubilin complex was not determined\",\n        \"Whether AMN itself contributes to ligand recognition was not resolved\"\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Human genetic analysis showed that AMN mutations in exons 1–4 cause Imerslund–Gräsbeck syndrome without embryonic lethality, resolving the paradox between the severe mouse phenotype and human disease by demonstrating alternative downstream translation initiation.\",\n      \"evidence\": \"Mutation screening in IGS patients, translation initiation site mapping\",\n      \"pmids\": [\"12590260\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Precise functional domains of the truncated AMN isoform were not mapped\",\n        \"Whether the shorter AMN isoform has reduced affinity for cubilin was not tested\"\n      ]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"In vivo chimera studies in adult mice showed that AMN-null tissues fail to localize cubilin to the cell surface and exhibit selective proteinuria of cubilin ligands, establishing AMN as essential for cubilin surface targeting across multiple polarized epithelia.\",\n      \"evidence\": \"ES cell↔blastocyst chimeras, immunolocalization of cubilin in kidney and yolk sac, urinary protein analysis\",\n      \"pmids\": [\"15342463\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The intracellular fate of cubilin in AMN-null cells (ER vs. degradation) was not determined at this stage\"\n      ]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Domain-mapping in polarized cells identified the cubilin EGF domains as the AMN-binding interface and showed AMN mediates ER export while cubilin contributes carbohydrate-dependent apical sorting signals, delineating the division of labor in cubam trafficking.\",\n      \"evidence\": \"Expression of cubilin and AMN domain fragments in MDCK cells, co-immunoprecipitation, tunicamycin treatment, apical vs. basolateral surface biotinylation\",\n      \"pmids\": [\"15976000\", \"15845892\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for the AMN–EGF domain interaction was not resolved\",\n        \"Identity of the ER quality-control checkpoint recognizing unpaired cubilin was not defined\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Silencing experiments in kidney cells revealed that both AMN and megalin are independently required for cubilin intracellular stability, establishing a tripartite complex (cubilin–AMN–megalin) at the renal brush border.\",\n      \"evidence\": \"Ligand-affinity chromatography and co-IP of renal brush-border membranes, siRNA silencing of megalin and AMN, cubilin half-life measurement\",\n      \"pmids\": [\"17990981\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether cubilin degradation upon AMN loss proceeds through proteasomal or lysosomal pathways was not determined\",\n        \"Relative contributions of AMN versus megalin to cubilin stability were not quantitatively separated\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identification of two redundant FXNPXF motifs in the AMN cytoplasmic tail that recruit Dab2 and ARH explained the molecular basis for clathrin-mediated cubam endocytosis.\",\n      \"evidence\": \"Sequential mutagenesis of AMN FXNPXF motifs, yeast two-hybrid interaction with Dab2 and ARH, functional endocytosis assays\",\n      \"pmids\": [\"20088845\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Relative in vivo contribution of Dab2 versus ARH to cubam endocytosis in different tissues was not resolved\",\n        \"Phosphorylation or other post-translational regulation of the FXNPXF motifs was not assessed\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Cross-species rescue of Drosophila nephrocyte protein uptake by human AMN demonstrated evolutionary conservation of AMN–cubilin co-receptor function, extending the paradigm beyond vertebrate polarized epithelia.\",\n      \"evidence\": \"Drosophila nephrocyte RNAi of dAMN, transgenic rescue with human AMN, protein uptake assays, electron microscopy\",\n      \"pmids\": [\"23264686\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether the AMN cytoplasmic endocytic signals are conserved in Drosophila was not tested\",\n        \"Ligand specificity of the fly cubam complex is poorly defined\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Absence of megalin protein in human terminal ileum despite cubilin–AMN co-expression showed that cubam can function megalin-independently for intestinal vitamin B12 absorption, resolving tissue-specific receptor composition.\",\n      \"evidence\": \"Immunohistochemistry and quantitative RT-PCR for LRP2 in adult human terminal ileum\",\n      \"pmids\": [\"25052491\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional endocytosis data confirming megalin-independence in ileum were not provided\",\n        \"Whether an alternative endocytic co-receptor substitutes for megalin in ileum is unknown\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Quantitative mass spectrometry and mutagenesis demonstrated that AMN-dependent N-linked glycosylation of cubilin—not merely physical association—is the critical step for ER-to-plasma-membrane trafficking, explaining how pathogenic missense mutations cause ER retention and disease.\",\n      \"evidence\": \"Quantitative membrane targeting assay, mass spectrometry of cubilin glycosylation sites, site-directed mutagenesis, patient-derived renal proximal tubular cell analysis\",\n      \"pmids\": [\"29402915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How AMN promotes cubilin glycosylation mechanistically (chaperone vs. conformation-enabling) is not resolved\",\n        \"Structural model of the AMN–cubilin interface at the ER remains unavailable\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis for AMN–cubilin interaction, the mechanism by which AMN facilitates cubilin glycosylation, and the signaling pathway downstream of AMN in visceral endoderm remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No high-resolution structure of the AMN–cubilin complex exists\",\n        \"The embryonic signaling function of AMN has not been linked to a specific ligand or pathway\",\n        \"Tissue-specific regulatory mechanisms controlling AMN expression are largely undefined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0038024\", \"supporting_discovery_ids\": [2, 8, 10]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 6, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 4, 5, 6, 12]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [6, 12]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 8, 10]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [6, 12]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [2, 3, 9]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"complexes\": [\n      \"cubam (cubilin–AMN)\"\n    ],\n    \"partners\": [\n      \"CUBN\",\n      \"LRP2\",\n      \"DAB2\",\n      \"LDLRAP1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}