{"gene":"EPB41L2","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":1998,"finding":"EPB41L2 (4.1G) encodes a 113-kDa protein with three conserved domains shared with 4.1R: a membrane-binding domain, a spectrin-actin binding domain, and a C-terminal domain. Different isoforms exhibit differential subcellular localizations, arising from both alternative splicing and distinct gene expression.","method":"cDNA cloning, sequence analysis, subcellular localization by isoform-specific expression","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 — foundational characterization with domain mapping and localization, single study","pmids":["9598318"],"is_preprint":false},{"year":2004,"finding":"4.1G binds the carboxyl-terminal domain of CD226 (PTA-1) and forms a dynamic molecular complex with human discs large (hDlg) in T cells; T cell stimulation induces PTA-1 and 4.1G to associate tightly with the cytoskeleton, and the PTA-1 C-terminal peptide influences which isoform of 4.1G is bound.","method":"Co-immunoprecipitation, cytoskeleton fractionation, domain truncation binding assays, T cell activation experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP with domain mapping, single lab","pmids":["15138281"],"is_preprint":false},{"year":2004,"finding":"4.1G binds via its C-terminal domain to the third intracellular loop of the A1 adenosine receptor (A1AR), inhibiting A1AR-mediated cAMP inhibition and intracellular calcium release, and altering cell-surface A1AR expression.","method":"Yeast two-hybrid screen, truncation binding studies, co-immunoprecipitation from brain tissue, functional cAMP and calcium assays in HEK-293 and CHO cells","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods: Y2H, Co-IP in tissue, functional assays with overexpression","pmids":["12974671"],"is_preprint":false},{"year":2004,"finding":"4.1G binds directly to the C-terminal tail of metabotropic glutamate receptor subtype 1α (mGlu1α), co-localizes with it in hippocampal neurons, increases mGlu1α ligand-binding ability, alters its cellular distribution, and influences mGlu1α-mediated cAMP accumulation.","method":"Co-immunoprecipitation from HEK293 cells and rat brain, domain truncation, immunofluorescence in hippocampal neurons, ligand binding assay, cAMP functional assay","journal":"Journal of neuroscience research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including tissue Co-IP, domain mapping, and functional assays","pmids":["15372499"],"is_preprint":false},{"year":2005,"finding":"4.1G interacts with the C-terminus of the PTH/PTH-related protein receptor (PTHR) and facilitates its cell-surface localization; full-length 4.1G (but not a dominant-negative C-terminal domain fragment) enhances PTHR-mediated ERK1/2 phosphorylation and intracellular calcium elevation.","method":"Yeast two-hybrid, co-localization by immunohistochemistry in COS-7 cells, cell-surface biotinylation assay, ERK phosphorylation assay, calcium measurement","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods with dominant-negative controls and functional readouts","pmids":["16029167"],"is_preprint":false},{"year":2006,"finding":"4.1G localizes to paranodal loops, Schmidt-Lanterman incisures (SLI), periaxonal, mesaxonal, and abaxonal membranes of Schwann cells in rodent sciatic nerve, with distribution shifting from diffuse in immature cells to discrete membrane domains during maturation.","method":"Immunohistochemistry, double immunolabeling, immunoelectron microscopy, Northern/Western blot analysis","journal":"Journal of neuroscience research","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization by immunoelectron microscopy with developmental context, single lab","pmids":["16752423"],"is_preprint":false},{"year":2007,"finding":"The C-terminal domain of 4.1G binds to the cytoplasmic tail of FcγRI (CD64); binding requires a membrane-proximal core motif HxxBxxxBB in FcγRI followed by hydrophobic and negatively charged residues, with an alternatively spliced 4.1G product showing increased binding.","method":"Yeast two-hybrid, alanine-scanning mutagenesis of FcγRI cytoplasmic tail","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 3 — Y2H with mutagenesis mapping, single lab","pmids":["18023480"],"is_preprint":false},{"year":2010,"finding":"4.1G binds to erythroid membrane proteins band 3, glycophorin C, CD44, p55, and calmodulin via its FERM/membrane-binding domain; Ca2+/calmodulin modulates these interactions, with the N-terminal headpiece region of 4.1G differentially affecting binding affinities for band 3 and glycophorin C compared to 4.1R135.","method":"In vitro binding assays, calmodulin-affinity binding, domain truncation analysis","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1–2 — in vitro reconstituted binding with domain mapping, single lab","pmids":["20812914"],"is_preprint":false},{"year":2011,"finding":"4.1G interacts with nectin-like 4 (NECL4) in testis Sertoli cells; deletion of 4.1G in mice leads to decreased NECL4 expression and altered NECL4 localization, impaired Sertoli-spermatogenic cell contact, and male infertility in B6-129 hybrid mice.","method":"4.1G knockout mouse generation, co-immunoprecipitation, immunolocalization, histology, ultrastructural electron microscopy","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — KO mouse with defined phenotype, Co-IP, and localization studies; multiple orthogonal methods","pmids":["21482674"],"is_preprint":false},{"year":2011,"finding":"4.1G is required to target MPP6 (membrane protein palmitoylated 6) to Schmidt-Lanterman incisures (SLI) in myelinated peripheral nerves; in 4.1G knockout mice MPP6 is mislocalized to cytoplasm near Schwann cell nuclei, and SLI shape is altered in aged knockouts.","method":"Co-immunoprecipitation, immunofluorescence in 4.1G knockout mice, immunoelectron microscopy","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — KO mouse with specific protein mislocalization phenotype, Co-IP, multiple imaging methods","pmids":["22025680"],"is_preprint":false},{"year":2011,"finding":"Serine phosphorylation of the FcγRI cytoplasmic domain (by CK2) promotes its interaction with protein 4.1G and targets FcγRI to lipid rafts; a non-phosphorylatable FcγRI mutant is excluded from lipid rafts.","method":"Yeast two-hybrid, co-immunoprecipitation from human PBMC, in vitro CK2 phosphorylation assay, immunostaining, lipid raft fractionation","journal":"Journal of leukocyte biology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods including in vitro phosphorylation, mutagenesis, and raft fractionation, single lab","pmids":["22003208"],"is_preprint":false},{"year":2012,"finding":"4.1G is required for normal organization of internodal proteins in peripheral myelinated nerves; deletion of 4.1G in Schwann cells leads to aberrant distribution of juxtaparanodal proteins (Kv1 channels, Caspr2, TAG-1) that pile up at juxtaparanodes rather than forming a double strand along internodes.","method":"4.1G knockout mouse, immunofluorescence, confocal microscopy of sciatic nerve","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — clean KO with defined protein mislocalization phenotype, multiple markers analyzed","pmids":["22291039"],"is_preprint":false},{"year":2012,"finding":"4.1G co-immunoprecipitates with spectrin and SERCA2 in cardiac muscle, with 4.1G localizing to intracellular structures coincident with sarcoplasmic reticulum.","method":"Co-immunoprecipitation, immunofluorescence, subcellular fractionation","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP with localization data, single lab","pmids":["22429617"],"is_preprint":false},{"year":2012,"finding":"Plasma membrane-associated 4.1G suppresses adenylyl cyclase-mediated cAMP production; this suppression requires the FERM domain for plasma membrane targeting, and is observed in membrane preparations of 4.1G-overexpressing cells.","method":"Overexpression, siRNA knockdown, FERM-domain deletion mutant, cAMP assays in HEK293 cells, membrane preparations","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 — gain/loss-of-function with domain mutant, in-cell and membrane fractionation assays, single lab","pmids":["23201780"],"is_preprint":false},{"year":2012,"finding":"4.1G FERM domain is essential for cellular arborization of oligodendrocyte cell line OLN-93; 4.1G promotes tight junction reassembly and its knockdown inhibits tight junction formation, with 4.1G clustering at cell periphery with ZO-1.","method":"Overexpression of domain-deleted constructs, siRNA knockdown, calcium switch tight junction assay, immunoprecipitation, immunofluorescence","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 — domain-deletion analysis with functional readouts, single lab","pmids":["21898413"],"is_preprint":false},{"year":2013,"finding":"4.1G interacts with a subset of CNG (cyclic-nucleotide gated) channels in rod outer segments (ROS) via its FERM and CTD domains; a smaller splice variant of 4.1G selectively binds CNG channels not associated with the peripherin-2-CNG channel complex.","method":"Immunoprecipitation, mass spectrometry, cDNA cloning, domain truncation binding assays, immunofluorescence","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 — IP/MS with domain-binding assays, single lab","pmids":["24144699"],"is_preprint":false},{"year":2013,"finding":"Src kinase is present in SLIs of sciatic nerves in a complex with MPP6 and 4.1G; in 4.1G-deficient nerves (lacking both 4.1G and MPP6 in SLIs), activated P418-Src immunoreactivity in SLIs is increased, and MPP6 co-immunoprecipitates with Src.","method":"Immunofluorescence with phospho-specific Src antibodies, co-immunoprecipitation, 4.1G knockout mouse comparison","journal":"Histochemistry and cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with KO comparison and phosphorylation state analysis, single lab","pmids":["23306908"],"is_preprint":false},{"year":2013,"finding":"Ca2+/calmodulin binding to the N-terminal headpiece region (GHP) of 4.1G — specifically to the peptide S71RGISRFIPPWLKKQKS — induces a conformational change from intrinsically disordered coiled structure to a compact structure, which sterically inhibits FERM domain interactions with membrane proteins.","method":"Small-angle X-ray scattering, NMR spectroscopy, circular dichroism, peptide binding assays","journal":"Cell biochemistry and biophysics","confidence":"High","confidence_rationale":"Tier 1 — multiple biophysical structural methods with peptide-level mechanistic resolution","pmids":["23354586"],"is_preprint":false},{"year":2015,"finding":"4.1G directly binds β1 integrin via its membrane-binding domain; in 4.1G knockout MEFs, surface expression of β1 integrin and its active form are reduced, focal adhesion kinase phosphorylation is suppressed, and cell adhesion, spreading, and migration are impaired.","method":"Co-immunoprecipitation, in vitro binding assay with domain-deleted constructs, cell-surface biotinylation, FAK phosphorylation assay, migration assay in 4.1G KO MEFs","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — KO cells with multiple orthogonal methods including in vitro binding and domain mapping","pmids":["26644476"],"is_preprint":false},{"year":2015,"finding":"4.1G binds to AP3B2 (adaptor protein 3 subunit β2, involved in neuronal membrane trafficking) and promotes neurite extension in an AP3B2-dependent manner; 4.1G-deficient retina shows mislocalization of photoreceptor terminals and impaired visual acuity.","method":"Co-immunoprecipitation, 4.1G knockout mouse, optokinetic response assay, immunofluorescence","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with in vivo KO phenotype, single lab","pmids":["25660028"],"is_preprint":false},{"year":2017,"finding":"4.1G is required for proper sorting of scaffold protein Lin7 (Lin7c and Lin7a) in sciatic nerves; 4.1G-deficient mice show loss of Lin7 immunolocalization and reduced Lin7 protein, and MPP6 co-immunoprecipitates with Lin7. Loss of 4.1G also causes myelin thickening and paranodal attachment defects.","method":"4.1G knockout mouse, immunofluorescence, co-immunoprecipitation, electron microscopy, motor conduction velocity measurement","journal":"Histochemistry and cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — KO with Co-IP and multiple phenotypic readouts, single lab","pmids":["28755316"],"is_preprint":false},{"year":2019,"finding":"4.1G directly binds adenylyl cyclase type 6 (AC6) via its FERM domain interacting with the N-terminus of AC6; this interaction suppresses AC6 activity at the plasma membrane, attenuating PTHR-mediated Gs/AC6/cAMP signaling. An AC6-N arginine-to-alanine mutant (AC6-N-3A) disrupts both 4.1G binding and plasma membrane distribution.","method":"Co-immunoprecipitation, in vitro GST pulldown binding assay, mutagenesis (AC6-N-3A), cAMP functional assay, 4.1G knockdown, dominant-negative AC6-N overexpression","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro direct binding reconstitution with mutagenesis and functional cAMP assay, multiple orthogonal approaches","pmids":["31383768"],"is_preprint":false},{"year":2022,"finding":"4.1G is required for primary ciliogenesis in preosteoblasts; 4.1G knockout mice show suppressed primary cilium formation and calcium deposits in trabecular bone, and 4.1G knockdown in MC3T3-E1 cells suppresses cilia elongation, Hedgehog signaling induction, and osteoblast differentiation.","method":"4.1G knockout mouse, siRNA knockdown, immunofluorescence (primary cilium markers), Hedgehog signaling assay, Alizarin Red calcium staining","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse and cell-based KD with defined pathway readout, single lab","pmids":["35216233"],"is_preprint":false},{"year":2023,"finding":"EPB41L2 (4.1G) interacts physically with Super-Conserved Receptors Expressed in the Brain (SREBs, orphan GPCRs); EPB41L2 co-localizes with SREB1 at the plasma membrane and its knockdown reduces plasma membrane localization of SREB1 and alters its detergent solubility, suggesting EPB41L2 regulates membrane microenvironment of these receptors.","method":"BioID2 proximity labeling, streptavidin pulldown with mass spectrometry, co-immunoprecipitation, immunofluorescence, siRNA knockdown","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 — proximity labeling confirmed by Co-IP and functional knockdown, single lab","pmids":["37998360"],"is_preprint":false},{"year":2025,"finding":"The C-terminal domain (CTD) of 4.1G is intrinsically disordered and forms a fuzzy complex with the disordered C-terminus of NuMA (nuclear mitotic apparatus protein); macromolecular crowding induces structural compaction of 4.1G-CTD while preserving its disordered character and enhances binding affinity and association kinetics with NuMA.","method":"NMR spectroscopy, small-angle X-ray scattering, circular dichroism, biophysical binding kinetics under crowding conditions","journal":"Physical chemistry chemical physics","confidence":"Medium","confidence_rationale":"Tier 1 — multiple biophysical methods, but single lab and no in-cell functional validation","pmids":["40726410"],"is_preprint":false}],"current_model":"EPB41L2 (4.1G) is a multifunctional membrane skeletal adaptor protein that, through its FERM/membrane-binding domain and C-terminal domain, links diverse transmembrane proteins (GPCRs including A1AR, mGlu1α, PTHR, SREBs; adhesion molecules NECL4, β1 integrin; ion channels CNG; immune receptors FcγRI, CD226) to the spectrin-actin cytoskeleton, thereby regulating their surface expression, subcellular localization, and downstream signaling — including direct suppression of adenylyl cyclase 6 activity to attenuate cAMP production — and is required in vivo for organization of myelinated nerve internodes, spermatogenesis, retinal synapse positioning, and primary ciliogenesis-dependent osteoblast differentiation, with Ca2+/calmodulin binding to its N-terminal headpiece providing a conformational regulatory switch that modulates FERM domain interactions."},"narrative":{"teleology":[{"year":1998,"claim":"Identification of EPB41L2 as a new 4.1 family member established that the membrane-binding, spectrin-actin binding, and C-terminal domains are conserved, and that alternative splicing generates isoforms with distinct subcellular distributions.","evidence":"cDNA cloning, sequence analysis, and isoform-specific localization","pmids":["9598318"],"confidence":"Medium","gaps":["No functional assays performed","Interacting partners unknown","Tissue-specific isoform functions uncharacterized"]},{"year":2004,"claim":"Discovery that 4.1G binds cytoplasmic tails of GPCRs (A1AR, mGlu1α) and the immune co-receptor CD226 revealed a general role as a GPCR/receptor scaffolding adaptor, with functional consequences for receptor surface expression and cAMP/calcium signaling.","evidence":"Yeast two-hybrid, Co-IP from brain tissue and transfected cells, domain truncation, cAMP and calcium functional assays, T cell activation and cytoskeleton fractionation","pmids":["12974671","15372499","15138281"],"confidence":"High","gaps":["Stoichiometry and affinity of GPCR interactions unknown","In vivo relevance of GPCR scaffolding not tested"]},{"year":2005,"claim":"Demonstration that 4.1G promotes PTHR surface localization and enhances downstream ERK and calcium signaling — blocked by a dominant-negative CTD fragment — established it as a positive regulator of receptor trafficking and signaling output.","evidence":"Y2H, cell-surface biotinylation, ERK phosphorylation and calcium assays with dominant-negative construct in COS-7 cells","pmids":["16029167"],"confidence":"High","gaps":["Mechanism of trafficking enhancement (recycling vs. degradation) unresolved","Dominant-negative effect not tested in vivo"]},{"year":2006,"claim":"Localization of 4.1G to specific Schwann cell membrane domains (paranodal loops, Schmidt-Lanterman incisures, periaxonal/abaxonal membranes) with developmental redistribution established it as a myelination-associated scaffold.","evidence":"Immunoelectron microscopy, double immunolabeling, and developmental Northern/Western blot analysis in rodent sciatic nerve","pmids":["16752423"],"confidence":"Medium","gaps":["No loss-of-function data at this stage","Binding partners in Schwann cell membranes unidentified"]},{"year":2010,"claim":"Biochemical reconstitution of FERM domain interactions with erythroid membrane proteins and demonstration that Ca²⁺/calmodulin modulates these interactions established 4.1G as a calcium-regulated membrane adaptor.","evidence":"In vitro binding assays with purified proteins, calmodulin-affinity binding, domain truncation analysis","pmids":["20812914"],"confidence":"Medium","gaps":["All data in vitro; in-cell calcium regulation not demonstrated","Relative affinities vs. 4.1R not functionally tested in cells"]},{"year":2011,"claim":"4.1G knockout mice revealed essential in vivo roles: NECL4-dependent Sertoli-spermatid adhesion for male fertility, and MPP6 targeting to Schmidt-Lanterman incisures for myelinated nerve organization, directly linking the adaptor function to tissue-level phenotypes.","evidence":"4.1G knockout mouse with histology, ultrastructural EM, Co-IP, immunolocalization in testis and sciatic nerve","pmids":["21482674","22025680"],"confidence":"High","gaps":["Whether fertility phenotype is strain-dependent remains uncertain","Direct vs. indirect role in MPP6 targeting not resolved"]},{"year":2012,"claim":"Multiple studies converged to show that 4.1G organizes internodal protein distribution in peripheral nerves, promotes tight junction assembly via its FERM domain, and suppresses adenylyl cyclase-mediated cAMP production at the plasma membrane, broadening its known functions beyond receptor scaffolding.","evidence":"4.1G KO sciatic nerve confocal microscopy (juxtaparanodal protein mislocalization), overexpression/siRNA with cAMP assays and FERM-deletion mutants, calcium switch tight junction assay in OLN-93 cells","pmids":["22291039","23201780","21898413"],"confidence":"High","gaps":["Identity of the adenylyl cyclase isoform targeted was unknown at this point","Tight junction function not validated in vivo"]},{"year":2013,"claim":"Structural biophysics revealed that Ca²⁺/calmodulin binding to the N-terminal headpiece induces a disorder-to-compact conformational transition that sterically occludes the FERM domain, providing a defined molecular switch mechanism for regulating 4.1G interactions.","evidence":"SAXS, NMR spectroscopy, and circular dichroism on purified headpiece and FERM domain constructs","pmids":["23354586"],"confidence":"High","gaps":["No in-cell validation of conformational switch","Physiological calcium concentrations triggering the switch not determined"]},{"year":2015,"claim":"Identification of β1 integrin as a direct FERM domain ligand, with KO MEFs showing reduced integrin surface expression and impaired adhesion/migration, and identification of AP3B2-dependent neurite extension with retinal synapse mislocalization in KO mice, extended 4.1G's roles to cell adhesion and neuronal morphogenesis.","evidence":"In vitro binding, Co-IP, surface biotinylation, FAK phosphorylation in KO MEFs; Co-IP with AP3B2, optokinetic response and retinal immunofluorescence in KO mice","pmids":["26644476","25660028"],"confidence":"High","gaps":["Whether integrin phenotype contributes to nerve or testis KO phenotypes untested","AP3B2 binding domain on 4.1G not mapped"]},{"year":2019,"claim":"Direct binding of the FERM domain to the N-terminus of adenylyl cyclase 6 was identified as the molecular basis for 4.1G-mediated cAMP suppression; mutagenesis of AC6 disrupted both binding and plasma membrane distribution, linking scaffold function to enzyme regulation.","evidence":"GST pulldown, Co-IP, AC6-N-3A mutagenesis, cAMP assay with 4.1G knockdown and dominant-negative AC6-N overexpression","pmids":["31383768"],"confidence":"High","gaps":["In vivo consequences of AC6 regulation by 4.1G not examined","Whether other AC isoforms are similarly regulated is unknown"]},{"year":2022,"claim":"Discovery that 4.1G is required for primary ciliogenesis and Hedgehog signaling in preosteoblasts, with KO mice showing bone mineralization defects, revealed an unexpected role in cilium-dependent developmental signaling.","evidence":"4.1G KO mouse bone phenotype, siRNA in MC3T3-E1 cells with cilium markers and Hedgehog pathway readouts, Alizarin Red staining","pmids":["35216233"],"confidence":"Medium","gaps":["Mechanism by which 4.1G promotes ciliogenesis is unknown","Whether ciliary defects extend to other tissues not examined","Single lab finding"]},{"year":2025,"claim":"Biophysical characterization showed that the intrinsically disordered CTD forms a fuzzy complex with NuMA, with macromolecular crowding enhancing binding, suggesting that cellular crowding conditions tune 4.1G scaffold interactions.","evidence":"NMR, SAXS, circular dichroism, and binding kinetics under crowding conditions with purified proteins","pmids":["40726410"],"confidence":"Medium","gaps":["No in-cell or functional validation of 4.1G-NuMA interaction","Physiological relevance of NuMA binding unknown","Whether crowding effects apply to other CTD partners untested"]},{"year":null,"claim":"Key open questions include the structural basis and selectivity rules governing how 4.1G discriminates among its many transmembrane partners, whether the Ca²⁺/calmodulin conformational switch operates in physiological signaling contexts in vivo, the mechanism by which 4.1G promotes primary ciliogenesis, and whether human disease phenotypes arise from EPB41L2 loss-of-function.","evidence":"","pmids":[],"confidence":"Low","gaps":["No human genetic disease linked to EPB41L2 mutations","No atomic-resolution structure of full-length 4.1G or FERM–partner complexes","In vivo calcium regulation of 4.1G conformational state untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,3,4,6,8,18,23]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,13,21]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,7,18]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[5,13,21,23]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,14]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3,4,13,21,22]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[3,5,9,11,15,19,20]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[8,14,18]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,6,10]}],"complexes":[],"partners":["NECL4","MPP6","AC6","A1AR","PTHR","ITGB1","CD226","NUMA"],"other_free_text":[]},"mechanistic_narrative":"EPB41L2 (protein 4.1G) is a membrane-skeletal adaptor that couples diverse transmembrane proteins to the cortical cytoskeleton, thereby controlling their surface expression, subcellular distribution, and downstream signaling in neural, immune, reproductive, and skeletal tissues. Its FERM domain anchors the protein to the plasma membrane and directly binds adenylyl cyclase 6 to suppress cAMP production, while its C-terminal domain engages the cytoplasmic tails of GPCRs (A1AR, mGlu1α, PTHR, SREBs), immune receptors (FcγRI, CD226), and adhesion molecules (β1 integrin, NECL4), modulating receptor-mediated ERK, calcium, and Hedgehog signaling [PMID:21482674, PMID:31383768, PMID:26644476, PMID:16029167]. Ca²⁺/calmodulin binding to the N-terminal headpiece induces a disorder-to-compact conformational switch that sterically inhibits FERM domain interactions, providing a calcium-dependent regulatory mechanism [PMID:23354586]. In vivo, 4.1G knockout mice display disorganized internodal protein distribution in peripheral myelin, male infertility from impaired Sertoli–spermatid contacts, mislocalized photoreceptor synaptic terminals with reduced visual acuity, and defective primary ciliogenesis in preosteoblasts [PMID:22291039, PMID:21482674, PMID:25660028, PMID:35216233]."},"prefetch_data":{"uniprot":{"accession":"O43491","full_name":"Band 4.1-like protein 2","aliases":["Erythrocyte membrane protein band 4.1-like 2","Generally expressed protein 4.1","4.1G"],"length_aa":1005,"mass_kda":112.6,"function":"Required for dynein-dynactin complex and NUMA1 recruitment at the mitotic cell cortex during anaphase (PubMed:23870127)","subcellular_location":"Cytoplasm, cytoskeleton; Cytoplasm, cell cortex; Cell membrane","url":"https://www.uniprot.org/uniprotkb/O43491/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EPB41L2","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CLNS1A","stoichiometry":0.2},{"gene":"NUMA1","stoichiometry":0.2},{"gene":"SRP9","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/EPB41L2","total_profiled":1310},"omim":[{"mim_id":"611804","title":"ELLIPTOCYTOSIS 1; EL1","url":"https://www.omim.org/entry/611804"},{"mim_id":"609744","title":"CELL ADHESION MOLECULE 4; CADM4","url":"https://www.omim.org/entry/609744"},{"mim_id":"603237","title":"ERYTHROCYTE MEMBRANE PROTEIN 4.1-LIKE 2; EPB41L2","url":"https://www.omim.org/entry/603237"},{"mim_id":"130500","title":"ERYTHROCYTE MEMBRANE PROTEIN BAND 4.1; EPB41","url":"https://www.omim.org/entry/130500"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Plasma membrane","reliability":"Enhanced"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Cell Junctions","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"retina","ntpm":327.6}],"url":"https://www.proteinatlas.org/search/EPB41L2"},"hgnc":{"alias_symbol":["4.1-G","4.1G"],"prev_symbol":[]},"alphafold":{"accession":"O43491","domains":[{"cath_id":"3.10.20.90","chopping":"216-297","consensus_level":"medium","plddt":93.7363,"start":216,"end":297},{"cath_id":"1.20.80.10","chopping":"298-403","consensus_level":"medium","plddt":93.6267,"start":298,"end":403},{"cath_id":"2.30.29.30","chopping":"407-503_513-531","consensus_level":"medium","plddt":90.8549,"start":407,"end":531},{"cath_id":"2.20.25","chopping":"922-962","consensus_level":"medium","plddt":73.1073,"start":922,"end":962}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43491","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43491-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43491-F1-predicted_aligned_error_v6.png","plddt_mean":59.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EPB41L2","jax_strain_url":"https://www.jax.org/strain/search?query=EPB41L2"},"sequence":{"accession":"O43491","fasta_url":"https://rest.uniprot.org/uniprotkb/O43491.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43491/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43491"}},"corpus_meta":[{"pmid":"9598318","id":"PMC_9598318","title":"Cloning and characterization of 4.1G (EPB41L2), a new member of the skeletal protein 4.1 (EPB41) gene family.","date":"1998","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/9598318","citation_count":97,"is_preprint":false},{"pmid":"15138281","id":"PMC_15138281","title":"The LFA-1-associated molecule PTA-1 (CD226) on T cells forms a dynamic molecular complex with protein 4.1G and human discs large.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15138281","citation_count":59,"is_preprint":false},{"pmid":"12974671","id":"PMC_12974671","title":"Cytoskeletal protein 4.1G binds to the third intracellular loop of the A1 adenosine receptor and inhibits receptor action.","date":"2004","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/12974671","citation_count":44,"is_preprint":false},{"pmid":"22291039","id":"PMC_22291039","title":"The cytoskeletal adapter protein 4.1G organizes the internodes in peripheral myelinated nerves.","date":"2012","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/22291039","citation_count":43,"is_preprint":false},{"pmid":"15372499","id":"PMC_15372499","title":"Cytoskeletal protein 4.1G is a binding partner of the metabotropic glutamate receptor subtype 1 alpha.","date":"2004","source":"Journal of neuroscience research","url":"https://pubmed.ncbi.nlm.nih.gov/15372499","citation_count":41,"is_preprint":false},{"pmid":"16029167","id":"PMC_16029167","title":"Increase in cell-surface localization of parathyroid hormone receptor by cytoskeletal protein 4.1G.","date":"2005","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/16029167","citation_count":30,"is_preprint":false},{"pmid":"21482674","id":"PMC_21482674","title":"Lack of protein 4.1G causes altered expression and localization of the cell adhesion molecule nectin-like 4 in testis and can cause male infertility.","date":"2011","source":"Molecular and cellular 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biology","url":"https://pubmed.ncbi.nlm.nih.gov/23306908","citation_count":16,"is_preprint":false},{"pmid":"32329852","id":"PMC_32329852","title":"CircRNA EPB41L2 inhibits tumorigenicity of lung adenocarcinoma through regulating CDH4 by miR-211-5p.","date":"2020","source":"European review for medical and pharmacological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32329852","citation_count":15,"is_preprint":false},{"pmid":"22429617","id":"PMC_22429617","title":"Isoforms of protein 4.1 are differentially distributed in heart muscle cells: relation of 4.1R and 4.1G to components of the Ca2+ homeostasis system.","date":"2012","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/22429617","citation_count":13,"is_preprint":false},{"pmid":"24144699","id":"PMC_24144699","title":"Interaction of 4.1G and cGMP-gated channels in rod photoreceptor outer segments.","date":"2013","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/24144699","citation_count":12,"is_preprint":false},{"pmid":"18023480","id":"PMC_18023480","title":"Protein 4.1G binds to a unique motif within the Fc gamma RI cytoplasmic tail.","date":"2007","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/18023480","citation_count":12,"is_preprint":false},{"pmid":"26644476","id":"PMC_26644476","title":"Protein 4.1G Regulates Cell Adhesion, Spreading, and Migration of Mouse Embryonic Fibroblasts through the β1 Integrin Pathway.","date":"2015","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26644476","citation_count":12,"is_preprint":false},{"pmid":"21904552","id":"PMC_21904552","title":"Insights into the Function of the Unstructured N-Terminal Domain of Proteins 4.1R and 4.1G in Erythropoiesis.","date":"2011","source":"International journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/21904552","citation_count":12,"is_preprint":false},{"pmid":"23201780","id":"PMC_23201780","title":"Suppression of adenylyl cyclase-mediated cAMP production by plasma membrane associated cytoskeletal protein 4.1G.","date":"2012","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/23201780","citation_count":10,"is_preprint":false},{"pmid":"22003208","id":"PMC_22003208","title":"Serine phosphorylation of FcγRI cytoplasmic domain directs lipid raft localization and interaction with protein 4.1G.","date":"2011","source":"Journal of leukocyte biology","url":"https://pubmed.ncbi.nlm.nih.gov/22003208","citation_count":10,"is_preprint":false},{"pmid":"20812914","id":"PMC_20812914","title":"Similarities and differences in the structure and function of 4.1G and 4.1R135, two protein 4.1 paralogues expressed in erythroid cells.","date":"2010","source":"The Biochemical 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Model.","date":"2020","source":"Nutrients","url":"https://pubmed.ncbi.nlm.nih.gov/32235804","citation_count":6,"is_preprint":false},{"pmid":"23354586","id":"PMC_23354586","title":"Novel mechanism of regulation of protein 4.1G binding properties through Ca2+/calmodulin-mediated structural changes.","date":"2013","source":"Cell biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/23354586","citation_count":5,"is_preprint":false},{"pmid":"21093541","id":"PMC_21093541","title":"Immunolocalization of membrane skeletal protein, 4.1G, in enteric glial cells in the mouse large intestine.","date":"2010","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/21093541","citation_count":5,"is_preprint":false},{"pmid":"21898413","id":"PMC_21898413","title":"4.1G promotes arborization and tight junction formation of oligodendrocyte cell line OLN-93.","date":"2012","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/21898413","citation_count":4,"is_preprint":false},{"pmid":"37998360","id":"PMC_37998360","title":"Proximity Interactome Analysis of Super Conserved Receptors Expressed in the Brain Identifies EPB41L2, SLC3A2, and LRBA as Main Partners.","date":"2023","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/37998360","citation_count":2,"is_preprint":false},{"pmid":"36178221","id":"PMC_36178221","title":"[Novel BRAF::EPB41L2 gene fusion in posterior fossa pilocytic astrocytoma. Brief communication].","date":"2022","source":"Arkhiv patologii","url":"https://pubmed.ncbi.nlm.nih.gov/36178221","citation_count":1,"is_preprint":false},{"pmid":"40726410","id":"PMC_40726410","title":"Exploring the effects of macromolecular crowding on the conformation and NuMA binding of 4.1G-CTD.","date":"2025","source":"Physical chemistry chemical physics : PCCP","url":"https://pubmed.ncbi.nlm.nih.gov/40726410","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17559,"output_tokens":5636,"usd":0.068609},"stage2":{"model":"claude-opus-4-6","input_tokens":9303,"output_tokens":3273,"usd":0.19251},"total_usd":0.261119,"stage1_batch_id":"msgbatch_0118RScg3WGaGkaZRtWZbtce","stage2_batch_id":"msgbatch_01Cpfzvtbj42aqp5oYZ5Te2s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"EPB41L2 (4.1G) encodes a 113-kDa protein with three conserved domains shared with 4.1R: a membrane-binding domain, a spectrin-actin binding domain, and a C-terminal domain. Different isoforms exhibit differential subcellular localizations, arising from both alternative splicing and distinct gene expression.\",\n      \"method\": \"cDNA cloning, sequence analysis, subcellular localization by isoform-specific expression\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — foundational characterization with domain mapping and localization, single study\",\n      \"pmids\": [\"9598318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"4.1G binds the carboxyl-terminal domain of CD226 (PTA-1) and forms a dynamic molecular complex with human discs large (hDlg) in T cells; T cell stimulation induces PTA-1 and 4.1G to associate tightly with the cytoskeleton, and the PTA-1 C-terminal peptide influences which isoform of 4.1G is bound.\",\n      \"method\": \"Co-immunoprecipitation, cytoskeleton fractionation, domain truncation binding assays, T cell activation experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with domain mapping, single lab\",\n      \"pmids\": [\"15138281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"4.1G binds via its C-terminal domain to the third intracellular loop of the A1 adenosine receptor (A1AR), inhibiting A1AR-mediated cAMP inhibition and intracellular calcium release, and altering cell-surface A1AR expression.\",\n      \"method\": \"Yeast two-hybrid screen, truncation binding studies, co-immunoprecipitation from brain tissue, functional cAMP and calcium assays in HEK-293 and CHO cells\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods: Y2H, Co-IP in tissue, functional assays with overexpression\",\n      \"pmids\": [\"12974671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"4.1G binds directly to the C-terminal tail of metabotropic glutamate receptor subtype 1α (mGlu1α), co-localizes with it in hippocampal neurons, increases mGlu1α ligand-binding ability, alters its cellular distribution, and influences mGlu1α-mediated cAMP accumulation.\",\n      \"method\": \"Co-immunoprecipitation from HEK293 cells and rat brain, domain truncation, immunofluorescence in hippocampal neurons, ligand binding assay, cAMP functional assay\",\n      \"journal\": \"Journal of neuroscience research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including tissue Co-IP, domain mapping, and functional assays\",\n      \"pmids\": [\"15372499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"4.1G interacts with the C-terminus of the PTH/PTH-related protein receptor (PTHR) and facilitates its cell-surface localization; full-length 4.1G (but not a dominant-negative C-terminal domain fragment) enhances PTHR-mediated ERK1/2 phosphorylation and intracellular calcium elevation.\",\n      \"method\": \"Yeast two-hybrid, co-localization by immunohistochemistry in COS-7 cells, cell-surface biotinylation assay, ERK phosphorylation assay, calcium measurement\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods with dominant-negative controls and functional readouts\",\n      \"pmids\": [\"16029167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"4.1G localizes to paranodal loops, Schmidt-Lanterman incisures (SLI), periaxonal, mesaxonal, and abaxonal membranes of Schwann cells in rodent sciatic nerve, with distribution shifting from diffuse in immature cells to discrete membrane domains during maturation.\",\n      \"method\": \"Immunohistochemistry, double immunolabeling, immunoelectron microscopy, Northern/Western blot analysis\",\n      \"journal\": \"Journal of neuroscience research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization by immunoelectron microscopy with developmental context, single lab\",\n      \"pmids\": [\"16752423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The C-terminal domain of 4.1G binds to the cytoplasmic tail of FcγRI (CD64); binding requires a membrane-proximal core motif HxxBxxxBB in FcγRI followed by hydrophobic and negatively charged residues, with an alternatively spliced 4.1G product showing increased binding.\",\n      \"method\": \"Yeast two-hybrid, alanine-scanning mutagenesis of FcγRI cytoplasmic tail\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Y2H with mutagenesis mapping, single lab\",\n      \"pmids\": [\"18023480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"4.1G binds to erythroid membrane proteins band 3, glycophorin C, CD44, p55, and calmodulin via its FERM/membrane-binding domain; Ca2+/calmodulin modulates these interactions, with the N-terminal headpiece region of 4.1G differentially affecting binding affinities for band 3 and glycophorin C compared to 4.1R135.\",\n      \"method\": \"In vitro binding assays, calmodulin-affinity binding, domain truncation analysis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro reconstituted binding with domain mapping, single lab\",\n      \"pmids\": [\"20812914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"4.1G interacts with nectin-like 4 (NECL4) in testis Sertoli cells; deletion of 4.1G in mice leads to decreased NECL4 expression and altered NECL4 localization, impaired Sertoli-spermatogenic cell contact, and male infertility in B6-129 hybrid mice.\",\n      \"method\": \"4.1G knockout mouse generation, co-immunoprecipitation, immunolocalization, histology, ultrastructural electron microscopy\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with defined phenotype, Co-IP, and localization studies; multiple orthogonal methods\",\n      \"pmids\": [\"21482674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"4.1G is required to target MPP6 (membrane protein palmitoylated 6) to Schmidt-Lanterman incisures (SLI) in myelinated peripheral nerves; in 4.1G knockout mice MPP6 is mislocalized to cytoplasm near Schwann cell nuclei, and SLI shape is altered in aged knockouts.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence in 4.1G knockout mice, immunoelectron microscopy\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with specific protein mislocalization phenotype, Co-IP, multiple imaging methods\",\n      \"pmids\": [\"22025680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Serine phosphorylation of the FcγRI cytoplasmic domain (by CK2) promotes its interaction with protein 4.1G and targets FcγRI to lipid rafts; a non-phosphorylatable FcγRI mutant is excluded from lipid rafts.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation from human PBMC, in vitro CK2 phosphorylation assay, immunostaining, lipid raft fractionation\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods including in vitro phosphorylation, mutagenesis, and raft fractionation, single lab\",\n      \"pmids\": [\"22003208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"4.1G is required for normal organization of internodal proteins in peripheral myelinated nerves; deletion of 4.1G in Schwann cells leads to aberrant distribution of juxtaparanodal proteins (Kv1 channels, Caspr2, TAG-1) that pile up at juxtaparanodes rather than forming a double strand along internodes.\",\n      \"method\": \"4.1G knockout mouse, immunofluorescence, confocal microscopy of sciatic nerve\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined protein mislocalization phenotype, multiple markers analyzed\",\n      \"pmids\": [\"22291039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"4.1G co-immunoprecipitates with spectrin and SERCA2 in cardiac muscle, with 4.1G localizing to intracellular structures coincident with sarcoplasmic reticulum.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, subcellular fractionation\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP with localization data, single lab\",\n      \"pmids\": [\"22429617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Plasma membrane-associated 4.1G suppresses adenylyl cyclase-mediated cAMP production; this suppression requires the FERM domain for plasma membrane targeting, and is observed in membrane preparations of 4.1G-overexpressing cells.\",\n      \"method\": \"Overexpression, siRNA knockdown, FERM-domain deletion mutant, cAMP assays in HEK293 cells, membrane preparations\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — gain/loss-of-function with domain mutant, in-cell and membrane fractionation assays, single lab\",\n      \"pmids\": [\"23201780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"4.1G FERM domain is essential for cellular arborization of oligodendrocyte cell line OLN-93; 4.1G promotes tight junction reassembly and its knockdown inhibits tight junction formation, with 4.1G clustering at cell periphery with ZO-1.\",\n      \"method\": \"Overexpression of domain-deleted constructs, siRNA knockdown, calcium switch tight junction assay, immunoprecipitation, immunofluorescence\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain-deletion analysis with functional readouts, single lab\",\n      \"pmids\": [\"21898413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"4.1G interacts with a subset of CNG (cyclic-nucleotide gated) channels in rod outer segments (ROS) via its FERM and CTD domains; a smaller splice variant of 4.1G selectively binds CNG channels not associated with the peripherin-2-CNG channel complex.\",\n      \"method\": \"Immunoprecipitation, mass spectrometry, cDNA cloning, domain truncation binding assays, immunofluorescence\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — IP/MS with domain-binding assays, single lab\",\n      \"pmids\": [\"24144699\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Src kinase is present in SLIs of sciatic nerves in a complex with MPP6 and 4.1G; in 4.1G-deficient nerves (lacking both 4.1G and MPP6 in SLIs), activated P418-Src immunoreactivity in SLIs is increased, and MPP6 co-immunoprecipitates with Src.\",\n      \"method\": \"Immunofluorescence with phospho-specific Src antibodies, co-immunoprecipitation, 4.1G knockout mouse comparison\",\n      \"journal\": \"Histochemistry and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with KO comparison and phosphorylation state analysis, single lab\",\n      \"pmids\": [\"23306908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Ca2+/calmodulin binding to the N-terminal headpiece region (GHP) of 4.1G — specifically to the peptide S71RGISRFIPPWLKKQKS — induces a conformational change from intrinsically disordered coiled structure to a compact structure, which sterically inhibits FERM domain interactions with membrane proteins.\",\n      \"method\": \"Small-angle X-ray scattering, NMR spectroscopy, circular dichroism, peptide binding assays\",\n      \"journal\": \"Cell biochemistry and biophysics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple biophysical structural methods with peptide-level mechanistic resolution\",\n      \"pmids\": [\"23354586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"4.1G directly binds β1 integrin via its membrane-binding domain; in 4.1G knockout MEFs, surface expression of β1 integrin and its active form are reduced, focal adhesion kinase phosphorylation is suppressed, and cell adhesion, spreading, and migration are impaired.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assay with domain-deleted constructs, cell-surface biotinylation, FAK phosphorylation assay, migration assay in 4.1G KO MEFs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO cells with multiple orthogonal methods including in vitro binding and domain mapping\",\n      \"pmids\": [\"26644476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"4.1G binds to AP3B2 (adaptor protein 3 subunit β2, involved in neuronal membrane trafficking) and promotes neurite extension in an AP3B2-dependent manner; 4.1G-deficient retina shows mislocalization of photoreceptor terminals and impaired visual acuity.\",\n      \"method\": \"Co-immunoprecipitation, 4.1G knockout mouse, optokinetic response assay, immunofluorescence\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with in vivo KO phenotype, single lab\",\n      \"pmids\": [\"25660028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"4.1G is required for proper sorting of scaffold protein Lin7 (Lin7c and Lin7a) in sciatic nerves; 4.1G-deficient mice show loss of Lin7 immunolocalization and reduced Lin7 protein, and MPP6 co-immunoprecipitates with Lin7. Loss of 4.1G also causes myelin thickening and paranodal attachment defects.\",\n      \"method\": \"4.1G knockout mouse, immunofluorescence, co-immunoprecipitation, electron microscopy, motor conduction velocity measurement\",\n      \"journal\": \"Histochemistry and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO with Co-IP and multiple phenotypic readouts, single lab\",\n      \"pmids\": [\"28755316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"4.1G directly binds adenylyl cyclase type 6 (AC6) via its FERM domain interacting with the N-terminus of AC6; this interaction suppresses AC6 activity at the plasma membrane, attenuating PTHR-mediated Gs/AC6/cAMP signaling. An AC6-N arginine-to-alanine mutant (AC6-N-3A) disrupts both 4.1G binding and plasma membrane distribution.\",\n      \"method\": \"Co-immunoprecipitation, in vitro GST pulldown binding assay, mutagenesis (AC6-N-3A), cAMP functional assay, 4.1G knockdown, dominant-negative AC6-N overexpression\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro direct binding reconstitution with mutagenesis and functional cAMP assay, multiple orthogonal approaches\",\n      \"pmids\": [\"31383768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"4.1G is required for primary ciliogenesis in preosteoblasts; 4.1G knockout mice show suppressed primary cilium formation and calcium deposits in trabecular bone, and 4.1G knockdown in MC3T3-E1 cells suppresses cilia elongation, Hedgehog signaling induction, and osteoblast differentiation.\",\n      \"method\": \"4.1G knockout mouse, siRNA knockdown, immunofluorescence (primary cilium markers), Hedgehog signaling assay, Alizarin Red calcium staining\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse and cell-based KD with defined pathway readout, single lab\",\n      \"pmids\": [\"35216233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EPB41L2 (4.1G) interacts physically with Super-Conserved Receptors Expressed in the Brain (SREBs, orphan GPCRs); EPB41L2 co-localizes with SREB1 at the plasma membrane and its knockdown reduces plasma membrane localization of SREB1 and alters its detergent solubility, suggesting EPB41L2 regulates membrane microenvironment of these receptors.\",\n      \"method\": \"BioID2 proximity labeling, streptavidin pulldown with mass spectrometry, co-immunoprecipitation, immunofluorescence, siRNA knockdown\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proximity labeling confirmed by Co-IP and functional knockdown, single lab\",\n      \"pmids\": [\"37998360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The C-terminal domain (CTD) of 4.1G is intrinsically disordered and forms a fuzzy complex with the disordered C-terminus of NuMA (nuclear mitotic apparatus protein); macromolecular crowding induces structural compaction of 4.1G-CTD while preserving its disordered character and enhances binding affinity and association kinetics with NuMA.\",\n      \"method\": \"NMR spectroscopy, small-angle X-ray scattering, circular dichroism, biophysical binding kinetics under crowding conditions\",\n      \"journal\": \"Physical chemistry chemical physics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — multiple biophysical methods, but single lab and no in-cell functional validation\",\n      \"pmids\": [\"40726410\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EPB41L2 (4.1G) is a multifunctional membrane skeletal adaptor protein that, through its FERM/membrane-binding domain and C-terminal domain, links diverse transmembrane proteins (GPCRs including A1AR, mGlu1α, PTHR, SREBs; adhesion molecules NECL4, β1 integrin; ion channels CNG; immune receptors FcγRI, CD226) to the spectrin-actin cytoskeleton, thereby regulating their surface expression, subcellular localization, and downstream signaling — including direct suppression of adenylyl cyclase 6 activity to attenuate cAMP production — and is required in vivo for organization of myelinated nerve internodes, spermatogenesis, retinal synapse positioning, and primary ciliogenesis-dependent osteoblast differentiation, with Ca2+/calmodulin binding to its N-terminal headpiece providing a conformational regulatory switch that modulates FERM domain interactions.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"EPB41L2 (protein 4.1G) is a membrane-skeletal adaptor that couples diverse transmembrane proteins to the cortical cytoskeleton, thereby controlling their surface expression, subcellular distribution, and downstream signaling in neural, immune, reproductive, and skeletal tissues. Its FERM domain anchors the protein to the plasma membrane and directly binds adenylyl cyclase 6 to suppress cAMP production, while its C-terminal domain engages the cytoplasmic tails of GPCRs (A1AR, mGlu1α, PTHR, SREBs), immune receptors (FcγRI, CD226), and adhesion molecules (β1 integrin, NECL4), modulating receptor-mediated ERK, calcium, and Hedgehog signaling [PMID:21482674, PMID:31383768, PMID:26644476, PMID:16029167]. Ca²⁺/calmodulin binding to the N-terminal headpiece induces a disorder-to-compact conformational switch that sterically inhibits FERM domain interactions, providing a calcium-dependent regulatory mechanism [PMID:23354586]. In vivo, 4.1G knockout mice display disorganized internodal protein distribution in peripheral myelin, male infertility from impaired Sertoli–spermatid contacts, mislocalized photoreceptor synaptic terminals with reduced visual acuity, and defective primary ciliogenesis in preosteoblasts [PMID:22291039, PMID:21482674, PMID:25660028, PMID:35216233].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Identification of EPB41L2 as a new 4.1 family member established that the membrane-binding, spectrin-actin binding, and C-terminal domains are conserved, and that alternative splicing generates isoforms with distinct subcellular distributions.\",\n      \"evidence\": \"cDNA cloning, sequence analysis, and isoform-specific localization\",\n      \"pmids\": [\"9598318\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional assays performed\", \"Interacting partners unknown\", \"Tissue-specific isoform functions uncharacterized\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Discovery that 4.1G binds cytoplasmic tails of GPCRs (A1AR, mGlu1α) and the immune co-receptor CD226 revealed a general role as a GPCR/receptor scaffolding adaptor, with functional consequences for receptor surface expression and cAMP/calcium signaling.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP from brain tissue and transfected cells, domain truncation, cAMP and calcium functional assays, T cell activation and cytoskeleton fractionation\",\n      \"pmids\": [\"12974671\", \"15372499\", \"15138281\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and affinity of GPCR interactions unknown\", \"In vivo relevance of GPCR scaffolding not tested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Demonstration that 4.1G promotes PTHR surface localization and enhances downstream ERK and calcium signaling — blocked by a dominant-negative CTD fragment — established it as a positive regulator of receptor trafficking and signaling output.\",\n      \"evidence\": \"Y2H, cell-surface biotinylation, ERK phosphorylation and calcium assays with dominant-negative construct in COS-7 cells\",\n      \"pmids\": [\"16029167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of trafficking enhancement (recycling vs. degradation) unresolved\", \"Dominant-negative effect not tested in vivo\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Localization of 4.1G to specific Schwann cell membrane domains (paranodal loops, Schmidt-Lanterman incisures, periaxonal/abaxonal membranes) with developmental redistribution established it as a myelination-associated scaffold.\",\n      \"evidence\": \"Immunoelectron microscopy, double immunolabeling, and developmental Northern/Western blot analysis in rodent sciatic nerve\",\n      \"pmids\": [\"16752423\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No loss-of-function data at this stage\", \"Binding partners in Schwann cell membranes unidentified\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Biochemical reconstitution of FERM domain interactions with erythroid membrane proteins and demonstration that Ca²⁺/calmodulin modulates these interactions established 4.1G as a calcium-regulated membrane adaptor.\",\n      \"evidence\": \"In vitro binding assays with purified proteins, calmodulin-affinity binding, domain truncation analysis\",\n      \"pmids\": [\"20812914\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"All data in vitro; in-cell calcium regulation not demonstrated\", \"Relative affinities vs. 4.1R not functionally tested in cells\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"4.1G knockout mice revealed essential in vivo roles: NECL4-dependent Sertoli-spermatid adhesion for male fertility, and MPP6 targeting to Schmidt-Lanterman incisures for myelinated nerve organization, directly linking the adaptor function to tissue-level phenotypes.\",\n      \"evidence\": \"4.1G knockout mouse with histology, ultrastructural EM, Co-IP, immunolocalization in testis and sciatic nerve\",\n      \"pmids\": [\"21482674\", \"22025680\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether fertility phenotype is strain-dependent remains uncertain\", \"Direct vs. indirect role in MPP6 targeting not resolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Multiple studies converged to show that 4.1G organizes internodal protein distribution in peripheral nerves, promotes tight junction assembly via its FERM domain, and suppresses adenylyl cyclase-mediated cAMP production at the plasma membrane, broadening its known functions beyond receptor scaffolding.\",\n      \"evidence\": \"4.1G KO sciatic nerve confocal microscopy (juxtaparanodal protein mislocalization), overexpression/siRNA with cAMP assays and FERM-deletion mutants, calcium switch tight junction assay in OLN-93 cells\",\n      \"pmids\": [\"22291039\", \"23201780\", \"21898413\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the adenylyl cyclase isoform targeted was unknown at this point\", \"Tight junction function not validated in vivo\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Structural biophysics revealed that Ca²⁺/calmodulin binding to the N-terminal headpiece induces a disorder-to-compact conformational transition that sterically occludes the FERM domain, providing a defined molecular switch mechanism for regulating 4.1G interactions.\",\n      \"evidence\": \"SAXS, NMR spectroscopy, and circular dichroism on purified headpiece and FERM domain constructs\",\n      \"pmids\": [\"23354586\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No in-cell validation of conformational switch\", \"Physiological calcium concentrations triggering the switch not determined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identification of β1 integrin as a direct FERM domain ligand, with KO MEFs showing reduced integrin surface expression and impaired adhesion/migration, and identification of AP3B2-dependent neurite extension with retinal synapse mislocalization in KO mice, extended 4.1G's roles to cell adhesion and neuronal morphogenesis.\",\n      \"evidence\": \"In vitro binding, Co-IP, surface biotinylation, FAK phosphorylation in KO MEFs; Co-IP with AP3B2, optokinetic response and retinal immunofluorescence in KO mice\",\n      \"pmids\": [\"26644476\", \"25660028\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether integrin phenotype contributes to nerve or testis KO phenotypes untested\", \"AP3B2 binding domain on 4.1G not mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Direct binding of the FERM domain to the N-terminus of adenylyl cyclase 6 was identified as the molecular basis for 4.1G-mediated cAMP suppression; mutagenesis of AC6 disrupted both binding and plasma membrane distribution, linking scaffold function to enzyme regulation.\",\n      \"evidence\": \"GST pulldown, Co-IP, AC6-N-3A mutagenesis, cAMP assay with 4.1G knockdown and dominant-negative AC6-N overexpression\",\n      \"pmids\": [\"31383768\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo consequences of AC6 regulation by 4.1G not examined\", \"Whether other AC isoforms are similarly regulated is unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Discovery that 4.1G is required for primary ciliogenesis and Hedgehog signaling in preosteoblasts, with KO mice showing bone mineralization defects, revealed an unexpected role in cilium-dependent developmental signaling.\",\n      \"evidence\": \"4.1G KO mouse bone phenotype, siRNA in MC3T3-E1 cells with cilium markers and Hedgehog pathway readouts, Alizarin Red staining\",\n      \"pmids\": [\"35216233\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which 4.1G promotes ciliogenesis is unknown\", \"Whether ciliary defects extend to other tissues not examined\", \"Single lab finding\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Biophysical characterization showed that the intrinsically disordered CTD forms a fuzzy complex with NuMA, with macromolecular crowding enhancing binding, suggesting that cellular crowding conditions tune 4.1G scaffold interactions.\",\n      \"evidence\": \"NMR, SAXS, circular dichroism, and binding kinetics under crowding conditions with purified proteins\",\n      \"pmids\": [\"40726410\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in-cell or functional validation of 4.1G-NuMA interaction\", \"Physiological relevance of NuMA binding unknown\", \"Whether crowding effects apply to other CTD partners untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the structural basis and selectivity rules governing how 4.1G discriminates among its many transmembrane partners, whether the Ca²⁺/calmodulin conformational switch operates in physiological signaling contexts in vivo, the mechanism by which 4.1G promotes primary ciliogenesis, and whether human disease phenotypes arise from EPB41L2 loss-of-function.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No human genetic disease linked to EPB41L2 mutations\", \"No atomic-resolution structure of full-length 4.1G or FERM–partner complexes\", \"In vivo calcium regulation of 4.1G conformational state untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 3, 4, 6, 8, 18, 23]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 13, 21]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 7, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 13, 21, 23]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 4, 13, 21, 22]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [3, 5, 9, 11, 15, 19, 20]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [8, 14, 18]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 6, 10]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NECL4\", \"MPP6\", \"AC6\", \"A1AR\", \"PTHR\", \"ITGB1\", \"CD226\", \"NuMA\"],\n    \"other_free_text\": []\n  }\n}\n```"}