{"gene":"EPB41L2","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":1998,"finding":"EPB41L2 (4.1G) encodes a 113-kDa protein with three regions of high homology to erythrocyte protein 4.1R: a membrane-binding domain, a spectrin-actin binding domain, and a C-terminal domain. Specific isoforms of 4.1G exhibit differential subcellular localizations, resulting from both alternative splicing and distinct gene expression.","method":"cDNA cloning, sequence analysis, subcellular localization studies","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cDNA cloning with sequence analysis and subcellular localization, single lab but multiple methods","pmids":["9598318"],"is_preprint":false},{"year":2004,"finding":"4.1G binds the carboxyl-terminal domain of the T cell adhesion molecule PTA-1 (CD226) and also associates with human discs large (hDlg). T cell stimulation causes PTA-1 and 4.1G to associate tightly with the cytoskeleton, and activated cells show altered binding of PTA-1 to the amino-terminal region of 4.1G, forming a dynamic molecular complex.","method":"Co-immunoprecipitation, membrane raft fractionation, cytoskeletal association assays, domain-binding studies","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with domain-mapping, single lab, multiple orthogonal methods","pmids":["15138281"],"is_preprint":false},{"year":2004,"finding":"4.1G binds to the third intracellular loop of the A1 adenosine receptor (A1AR) via its C-terminal domain. This interaction was confirmed in brain tissue and in HEK-293 and CHO cells. 4.1G overexpression reduced A1AR-mediated inhibition of cAMP accumulation, intracellular calcium release, and altered cell-surface A1AR expression.","method":"Yeast two-hybrid screening, truncation binding studies, co-immunoprecipitation in brain tissue, functional cAMP and calcium assays in HEK-293 and CHO cells","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid identification, confirmed by Co-IP in native tissue, functional readouts with multiple cell lines, multiple orthogonal methods","pmids":["12974671"],"is_preprint":false},{"year":2004,"finding":"4.1G directly interacts with the metabotropic glutamate receptor subtype 1alpha (mGlu1alpha) via the C-terminal tail of mGlu1alpha, co-localizes with mGlu1alpha in hippocampal neurons, and modulates mGlu1alpha-mediated cAMP accumulation, ligand-binding ability, and cellular distribution.","method":"Co-localization in hippocampal neurons, co-immunoprecipitation in HEK-293 cells and rat brain tissue, domain truncation analysis, functional cAMP assays","journal":"Journal of neuroscience research","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP in native brain tissue and transfected cells, co-localization in neurons, functional assays, multiple orthogonal methods","pmids":["15372499"],"is_preprint":false},{"year":2005,"finding":"4.1G interacts with the C-terminus of the parathyroid hormone receptor (PTHR) and facilitates cell-surface localization of PTHR, as shown by cell-surface biotinylation. The full-length 4.1G (but not 4.1G-CTD dominant-negative) enhanced PTH-stimulated ERK1/2 phosphorylation and intracellular Ca2+ elevation.","method":"Yeast two-hybrid, co-localization in COS-7 cells, cell-surface biotinylation assay, ERK phosphorylation and Ca2+ assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid, cell-surface biotinylation with dominant-negative controls, functional signaling assays, multiple orthogonal methods","pmids":["16029167"],"is_preprint":false},{"year":2006,"finding":"4.1G is expressed in Schwann cells of the peripheral nervous system and is specifically localized at paranodal loops, Schmidt-Lanterman incisures, and periaxonal, mesaxonal, and abaxonal membranes. During development, 4.1G transitions from diffuse distribution in immature Schwann cells to discrete localization at these membrane specializations during myelination.","method":"Northern blot, Western blot, immunohistochemistry with specific antibody, double immunolabeling, immunoelectron microscopy","journal":"Journal of neuroscience research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple immunolabeling methods including electron microscopy, single lab","pmids":["16752423"],"is_preprint":false},{"year":2007,"finding":"The C-terminal domain of 4.1G interacts with the cytoplasmic tail of FcγRI (CD64). A specific Fc gamma RI membrane-proximal core motif of HxxBxxxBB followed by hydrophobic and charged residues is central for 4.1G interaction, identified by Fc gamma RI truncation and alanine-substitution mutant analysis.","method":"Yeast two-hybrid, domain truncation analysis, alanine substitution mutagenesis","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid with systematic mutagenesis, single lab","pmids":["18023480"],"is_preprint":false},{"year":2009,"finding":"Mice with deletion of 4.1G and knockdown of 4.1N to ~22% of wild-type levels (combined ~12% hippocampal expression) showed a moderate reduction in synaptosomal GluR1 at 3 weeks of age, but no change in basic glutamatergic synaptic transmission or long-term potentiation, indicating 4.1G and 4.1N do not have a crucial role in glutamatergic synaptic transmission.","method":"Knockout and knockdown mouse model, electrophysiology, synaptosomal fractionation and immunoblotting","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO combined with electrophysiology and biochemical fractionation; this is a negative result experimentally established with rigorous controls","pmids":["19225127"],"is_preprint":false},{"year":2010,"finding":"4.1G associates with cell adhesion molecule-1 (CADM1) in seminiferous tubule lysates, as shown by co-immunoprecipitation. 4.1G is immunolocalized along cell membranes of Sertoli cells, spermatogonia, and early spermatocytes, and is expressed in spermatogonial stem cells at cell-cell contact regions.","method":"Immunolocalization, immunoprecipitation, in vitro spermatogonial stem cell culture, immunoblotting","journal":"Reproduction (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP in native tissue, confirmed localization by multiple methods, single lab","pmids":["20200204"],"is_preprint":false},{"year":2010,"finding":"4.1G binds erythroid membrane proteins including band 3, glycophorin C, CD44, and p55 via its membrane-binding domain. The N-terminal headpiece region of 4.1G differentiates its binding affinities from those of 4.1R135 for band 3 and glycophorin C. The headpiece also contains a high-affinity calcium-dependent calmodulin-binding site that modulates interactions with these membrane proteins.","method":"In vitro binding assays, affinity characterization, calmodulin interaction studies","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstituted binding assays with defined domain constructs and calmodulin regulation, single lab with multiple orthogonal methods","pmids":["20812914"],"is_preprint":false},{"year":2011,"finding":"4.1G deficiency in mice (B6-129 hybrid background) causes male infertility associated with atrophy, impaired cell-cell contact, and sloughing of spermatogenic cells. 4.1G associates with NECL4 (nectin-like 4) in Sertoli cells, and NECL4 expression is decreased and mislocalized in 4.1G-/- testis.","method":"Knockout mouse model, histology, ultrastructural analysis (electron microscopy), co-immunoprecipitation, immunolocalization","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with defined infertility phenotype, EM ultrastructure, Co-IP, and immunolocalization, multiple orthogonal methods","pmids":["21482674"],"is_preprint":false},{"year":2011,"finding":"4.1G co-localizes with MPP6 at Schmidt-Lanterman incisures and paranodes in sciatic nerve. MPP6 co-immunoprecipitates with 4.1G. In 4.1G knockout mice, MPP6 is mislocalized to the cytoplasm near Schwann cell nuclei, demonstrating that 4.1G is required for targeting MPP6 to Schmidt-Lanterman incisures.","method":"Immunofluorescence co-localization, co-immunoprecipitation, 4.1G knockout mouse analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP in native tissue confirmed with KO mouse showing specific mislocalization, multiple orthogonal methods","pmids":["22025680"],"is_preprint":false},{"year":2011,"finding":"In heart muscle cells, 4.1G is localized to intracellular structures coincident with sarcoplasmic reticulum and exists in an immunoprecipitable complex with spectrin and SERCA2.","method":"Immunofluorescence, immunoprecipitation, subcellular fractionation","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and subcellular fractionation showing complex with SERCA2, single lab","pmids":["22429617"],"is_preprint":false},{"year":2011,"finding":"Serine phosphorylation of the FcγRI cytoplasmic tail by CK2 promotes preferential interaction with protein 4.1G in vitro. 4.1G co-localizes with FcγRI in unstimulated U937 cells where CY is constitutively serine-phosphorylated; FcγRI cross-linking causes uncoupling. A nonphosphorylatable FcγRI mutant is excluded from lipid rafts, implicating 4.1G in phosphoserine-dependent targeting of FcγRI to lipid rafts.","method":"Yeast two-hybrid, in vitro binding with CK2-phosphorylated peptides, co-immunoprecipitation in human PBMC, immunostaining, lipid raft fractionation, mutagenesis","journal":"Journal of leukocyte biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro phosphorylation-dependent binding with mutagenesis, confirmed by Co-IP in primary cells and lipid raft fractionation, multiple orthogonal methods","pmids":["22003208"],"is_preprint":false},{"year":2012,"finding":"Deletion of 4.1G in Schwann cells causes aberrant distribution of internodal proteins including juxtaparanodal Kv1 channels, Caspr2, and TAG-1, and paranodal junction components. In 4.1G-/- mice, these proteins aggregate at the juxtaparanodal region rather than forming the normal double strand flanking paranodal junction components along internodes.","method":"4.1G knockout mouse, immunofluorescence, confocal microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with defined molecular phenotype in axoglial organization, replicated across multiple protein markers","pmids":["22291039"],"is_preprint":false},{"year":2012,"finding":"4.1G overexpression suppresses forskolin-induced and PTH-stimulated cAMP production in HEK293 cells; 4.1G knockdown increases cAMP production. A FERM-domain-deleted 4.1G mutant lacking plasma membrane distribution does not alter cAMP production, indicating that plasma membrane association of 4.1G is required for its suppression of adenylyl cyclase activity.","method":"Overexpression and siRNA knockdown in HEK293 cells, cAMP assay, membrane fractionation, FERM deletion mutagenesis","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 / Strong — gain- and loss-of-function with domain mutagenesis and membrane fractionation, multiple orthogonal methods in single lab","pmids":["23201780"],"is_preprint":false},{"year":2012,"finding":"4.1G overexpression promotes arborization of oligodendrocyte cell line OLN-93 through its FERM domain, while FERM-domain-deleted 4.1G does not. 4.1G also promotes tight junction reassembly (shown by calcium switch experiment) and its knockdown inhibits tight junction formation, with 4.1G co-clustering with ZO-1 at cell periphery.","method":"Overexpression and siRNA knockdown in OLN-93 cells, calcium switch assay, immunoprecipitation, immunofluorescence, domain deletion analysis","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with domain mutagenesis and functional readouts, single lab","pmids":["21898413"],"is_preprint":false},{"year":2013,"finding":"4.1G interacts with a subset of CNG channels in rod outer segments (ROS) through its FERM and CTD domains, identified by immunoprecipitation/mass spectrometry and confirmed by truncation and domain-binding assays. A smaller splice variant of 4.1G selectively interacted with CNG channels not associated with the peripherin-2-CNG channel complex.","method":"Immunoprecipitation and mass spectrometry, domain truncation analysis, domain-binding assays, immunofluorescence","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — IP-MS identification followed by systematic domain truncation and binding assays, multiple orthogonal methods","pmids":["24144699"],"is_preprint":false},{"year":2013,"finding":"Src kinase is present in Schmidt-Lanterman incisures and forms a complex with MPP6. In 4.1G-deficient nerve fibers (which lack both 4.1G and MPP6 from SLIs), active (P418) Src immunoreactivity in SLIs is enhanced compared to wild-type, implicating the 4.1G-MPP6 complex in restraining Src activity at SLIs.","method":"Immunostaining with phospho-specific antibodies, 4.1G knockout mouse analysis, immunoprecipitation","journal":"Histochemistry and cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP of Src-MPP6 plus KO phenotype analysis, single lab","pmids":["23306908"],"is_preprint":false},{"year":2013,"finding":"Ca2+/calmodulin binds to the N-terminal headpiece region (GHP) of 4.1G at the peptide SRGISRFIPPWLKKQKS, inducing a conformational switch from intrinsically disordered coiled structure to compact structure. This structural change sterically inhibits 4.1G FERM domain interactions with membrane proteins.","method":"Small-angle X-ray scattering, NMR spectroscopy, circular dichroism spectroscopy, peptide binding assays","journal":"Cell biochemistry and biophysics","confidence":"High","confidence_rationale":"Tier 1 / Moderate — three orthogonal structural biophysical methods identifying the Ca2+/CaM binding site and conformational mechanism, single lab","pmids":["23354586"],"is_preprint":false},{"year":2015,"finding":"4.1G binds directly to β1 integrin via its membrane-binding domain (shown by Co-IP and in vitro binding assays). In 4.1G-/- mouse embryonic fibroblasts, cell surface expression of β1 integrin and its active form are decreased, adhesion, spreading, and migration are impaired, and focal adhesion kinase phosphorylation is suppressed.","method":"4.1G knockout MEF cells, co-immunoprecipitation, in vitro binding assay, cell-surface FACS, migration assays, FAK phosphorylation analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct in vitro binding assay confirmed with KO cells showing surface integrin reduction, downstream signaling changes, and functional phenotype; multiple orthogonal methods","pmids":["26644476"],"is_preprint":false},{"year":2015,"finding":"4.1G is highly expressed in retinal photoreceptors and binds to AP3B2 (a protein involved in neuronal membrane trafficking). 4.1G-deficient retinas show mislocalization of photoreceptor terminals (without loss of synaptic connections), and 4.1G promotes neurite extension in an AP3B2-dependent manner. 4.1G mutant mice show visual acuity impairment.","method":"4.1G KO mouse, protein interaction (binding assay), immunohistochemistry, optokinetic response test, neurite extension assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with defined anatomical phenotype, binding partner identification, functional rescue/dependency demonstrated with AP3B2, multiple orthogonal methods","pmids":["25660028"],"is_preprint":false},{"year":2017,"finding":"4.1G deficiency in mice causes myelin abnormalities in the peripheral nervous system (thicker myelin internodes, distorted paranodal tips), slowed motor-conduction velocity, and loss of Lin7c and Lin7a (scaffold proteins) from sciatic nerves. MPP6 interacts with Lin7 by immunoprecipitation, and 4.1G is required for proper Lin7 sorting in Schwann cells.","method":"4.1G-/- mouse model, electron microscopy, electrophysiology, immunoprecipitation, immunofluorescence","journal":"Histochemistry and cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with EM, electrophysiology, Co-IP showing MPP6-Lin7 interaction, multiple orthogonal methods","pmids":["28755316"],"is_preprint":false},{"year":2019,"finding":"4.1G directly and selectively binds to the N-terminus of adenylyl cyclase type 6 (AC6) via its FERM domain, as shown by in vitro binding assays. Three consecutive arginine residues in AC6-N are required for 4.1G-FERM binding and for proper plasma membrane distribution of AC6. This interaction suppresses AC6 catalytic activity, attenuating PTHR-mediated Gs/AC6/cAMP signaling.","method":"Co-immunoprecipitation, in vitro binding assay, site-directed mutagenesis (AC6-N-3A), competitive inhibition with AC6-N overexpression, siRNA knockdown, cAMP assay","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro direct binding with mutagenesis, confirmed by Co-IP in cells, competitive inhibitor approach and knockdown, multiple rigorous methods","pmids":["31383768"],"is_preprint":false},{"year":2022,"finding":"4.1G is expressed in bone and is required for primary ciliogenesis and osteoblast differentiation. In 4.1G-knockout mice, calcium deposits and primary cilium formation are suppressed in preosteoblast-rich trabecular bone. Knockdown of 4.1G in MC3T3-E1 cells suppresses cilium elongation and inhibits cilia-mediated Hedgehog signaling and subsequent osteoblast differentiation.","method":"4.1G KO mouse, siRNA knockdown in MC3T3-E1 cells, immunofluorescence, calcium deposition assay, Hedgehog signaling assay","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse phenotype confirmed by cell-based knockdown with defined molecular pathway (Hedgehog), single lab","pmids":["35216233"],"is_preprint":false},{"year":2023,"finding":"EPB41L2 (4.1G) is a proximity interactor of Super-Conserved Receptors Expressed in the Brain (SREBs), confirmed by BioID2 proximity labeling and co-immunoprecipitation. EPB41L2 promotes plasma membrane localization of SREB1 and modifies SREB1 membrane microenvironment (increased detergent solubilization) as shown by siRNA knockdown.","method":"BioID2 proximity labeling, mass spectrometry, co-immunoprecipitation, siRNA knockdown, immunofluorescence","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity labeling with MS confirmed by Co-IP, 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 disorder, enhances binding affinity for NuMA, and accelerates association kinetics.","method":"Small-angle X-ray scattering, NMR spectroscopy, biophysical binding assays under crowding conditions","journal":"Physical chemistry chemical physics : PCCP","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple structural biophysical methods (SAXS, NMR) with binding kinetics analysis; interaction with NuMA established in vitro","pmids":["40726410"],"is_preprint":false}],"current_model":"EPB41L2 (4.1G) is a multidomain membrane skeletal adaptor protein whose FERM domain anchors it to the plasma membrane and mediates direct binding to diverse transmembrane proteins—including GPCRs (A1AR, mGlu1α, PTHR, SREB1), integrins (β1), ion channels (CNG channels, adenylyl cyclase AC6), and immune receptors (FcγRI, PTA-1/CD226)—while its CTD interacts with NuMA and scaffolding proteins (hDlg, MPP6, Lin7); through these interactions 4.1G regulates cell-surface localization and signaling of its binding partners (promoting receptor surface expression, suppressing AC6/cAMP output via direct FERM-AC6N binding), organizes internodal and Schmidt-Lanterman incisure architecture in Schwann cells by targeting MPP6 and restraining Src activity, supports spermatogenic cell adhesion via NECL4, drives photoreceptor synapse positioning via AP3B2-dependent membrane trafficking, facilitates cell adhesion and migration by maintaining surface-active β1 integrin, and promotes primary ciliogenesis and Hedgehog-mediated osteoblast differentiation; calmodulin binding to the N-terminal headpiece induces a disorder-to-order transition that allosterically inhibits FERM domain interactions, providing Ca2+-dependent regulation of the protein's scaffolding activity."},"narrative":{"mechanistic_narrative":"EPB41L2 (4.1G) is a multidomain membrane-skeletal adaptor that organizes the cell surface by linking transmembrane proteins to the underlying cytoskeleton, built from a membrane-binding (FERM) domain, a spectrin-actin binding domain, and a C-terminal domain (CTD) [PMID:9598318]. Through its FERM domain it docks directly to diverse membrane partners—β1 integrin [PMID:26644476] and the N-terminus of adenylyl cyclase type 6 (AC6) via a triple-arginine motif [PMID:31383768]—while its CTD engages G-protein-coupled receptors including the A1 adenosine receptor [PMID:12974671], mGlu1α [PMID:15372499], and the parathyroid hormone receptor [PMID:16029167], as well as the immune receptor FcγRI [PMID:18023480] and CNG channels of photoreceptors [PMID:24144699]. By controlling the cell-surface delivery and microenvironment of these partners, 4.1G tunes their signaling output: it promotes surface expression of PTHR and β1 integrin [PMID:16029167, PMID:26644476] yet directly suppresses AC6 catalytic activity to dampen Gs/cAMP signaling, an effect requiring FERM-dependent plasma-membrane association [PMID:23201780, PMID:31383768]. In Schwann cells 4.1G localizes to paranodes and Schmidt-Lanterman incisures and is required to target the scaffold MPP6 and Lin7 proteins, organize internodal channel distribution, and restrain Src activity, with its loss causing myelin abnormalities and slowed nerve conduction [PMID:22025680, PMID:22291039, PMID:23306908, PMID:28755316]. 4.1G also supports spermatogenic cell adhesion through NECL4 (loss causes male infertility) [PMID:21482674, PMID:22025680], positions photoreceptor synaptic terminals via AP3B2-dependent trafficking [PMID:25660028], and promotes primary ciliogenesis and Hedgehog-driven osteoblast differentiation [PMID:35216233]. Its scaffolding is allosterically gated: Ca2+/calmodulin binding to the N-terminal headpiece drives a disorder-to-order transition that sterically inhibits FERM-domain interactions, providing Ca2+-dependent regulation [PMID:20812914, PMID:23354586], while the intrinsically disordered CTD forms a fuzzy, crowding-sensitive complex with NuMA [PMID:40726410].","teleology":[{"year":1998,"claim":"Established 4.1G as a protein 4.1R paralog with a conserved tripartite domain architecture, framing it as a candidate membrane-skeletal adaptor with isoform-specific localization.","evidence":"cDNA cloning, sequence analysis, and subcellular localization of isoforms","pmids":["9598318"],"confidence":"Medium","gaps":["No binding partners or function identified","Isoform-specific roles not assigned"]},{"year":2004,"claim":"Demonstrated that 4.1G acts as a receptor-anchoring scaffold by binding GPCR intracellular domains (A1AR, mGlu1α) and the adhesion molecule PTA-1/CD226, modulating receptor surface expression and downstream cAMP/Ca2+ signaling.","evidence":"Yeast two-hybrid, Co-IP in brain tissue and transfected cells, functional cAMP/Ca2+ assays","pmids":["15138281","12974671","15372499"],"confidence":"High","gaps":["Domain responsible for receptor binding not fully delineated across partners","Whether 4.1G acts in receptor trafficking vs. retention unclear"]},{"year":2005,"claim":"Showed 4.1G facilitates cell-surface localization of a GPCR (PTHR) and amplifies its downstream signaling, requiring full-length protein, establishing a positive trafficking/scaffolding role distinct from signal suppression.","evidence":"Yeast two-hybrid, cell-surface biotinylation with CTD dominant-negative, ERK and Ca2+ assays in COS-7","pmids":["16029167"],"confidence":"High","gaps":["Mechanism of surface delivery (trafficking vs. stabilization) not resolved","Not validated in native tissue"]},{"year":2007,"claim":"Mapped a defined membrane-proximal motif (HxxBxxxBB) in FcγRI required for CTD binding, providing a sequence basis for 4.1G recognition of immune-receptor tails.","evidence":"Yeast two-hybrid, truncation and alanine-scanning mutagenesis","pmids":["18023480"],"confidence":"Medium","gaps":["Interaction not confirmed in primary immune cells at this stage","Functional consequence not yet shown"]},{"year":2009,"claim":"Genetic loss-of-function established that 4.1G (with 4.1N) is not essential for glutamatergic synaptic transmission or LTP, bounding its in vivo role in the hippocampus despite GluR1 changes.","evidence":"4.1G KO / 4.1N knockdown mice, electrophysiology, synaptosomal immunoblotting","pmids":["19225127"],"confidence":"High","gaps":["Redundancy with other 4.1 family members not fully excluded","Subtle synaptic phenotypes not probed"]},{"year":2010,"claim":"Defined 4.1G's membrane-binding domain interactions with classical erythroid skeletal partners and adhesion molecules, and identified a Ca2+-dependent calmodulin site in the headpiece that modulates these interactions—first hint of allosteric regulation.","evidence":"In vitro binding/affinity assays, calmodulin interaction studies; Co-IP and immunolocalization in seminiferous tubules (CADM1)","pmids":["20812914","20200204"],"confidence":"High","gaps":["Structural basis of CaM regulation not yet defined","Physiological context of membrane-protein binding incomplete"]},{"year":2011,"claim":"In vivo KO models established 4.1G as essential for organizing membrane specializations: it targets MPP6 to Schmidt-Lanterman incisures in Schwann cells and supports spermatogenic adhesion via NECL4, with loss causing male infertility.","evidence":"4.1G KO mice, EM ultrastructure, Co-IP, immunolocalization in testis and sciatic nerve; SERCA2/spectrin complex in heart","pmids":["21482674","22025680","22429617"],"confidence":"High","gaps":["Mechanism linking MPP6 mislocalization to myelin defects not yet defined","SERCA2 complex functional role uncharacterized"]},{"year":2012,"claim":"Resolved the mechanistic basis of 4.1G-mediated signal suppression and membrane organization—FERM-dependent plasma-membrane association is required to suppress adenylyl cyclase/cAMP output and to organize internodal channels (Kv1, Caspr2, TAG-1) and tight junctions.","evidence":"Gain/loss of function with FERM-deletion mutants, cAMP assays, KO mouse internodal marker imaging, calcium-switch tight-junction assays","pmids":["23201780","22291039","21898413"],"confidence":"High","gaps":["The adenylyl cyclase isoform directly bound not yet identified at this stage","Link between membrane scaffolding and channel clustering mechanism unclear"]},{"year":2013,"claim":"Extended the partner repertoire to photoreceptor CNG channels and demonstrated phosphoserine-dependent partner selection (CK2-phosphorylated FcγRI) and a role in restraining Src at incisures, revealing post-translational and signaling control of 4.1G scaffolding.","evidence":"IP-MS, domain-binding assays, in vitro CK2-phosphopeptide binding, Co-IP in primary cells, lipid-raft fractionation, KO Src phospho-imaging","pmids":["24144699","22003208","23306908"],"confidence":"High","gaps":["How phosphorylation switches partner affinity structurally not resolved","Mechanism by which 4.1G-MPP6 restrains Src unknown"]},{"year":2013,"claim":"Provided the structural mechanism for Ca2+ regulation: calmodulin binding to a defined headpiece peptide drives a disorder-to-order transition that sterically inhibits FERM-domain membrane interactions.","evidence":"SAXS, NMR, circular dichroism, peptide binding assays","pmids":["23354586"],"confidence":"High","gaps":["Regulation not demonstrated for full-length protein in cells","Which membrane partners are most sensitive to CaM gating untested"]},{"year":2015,"claim":"KO-based studies established 4.1G as a determinant of β1-integrin surface availability and adhesion/migration, and of photoreceptor terminal positioning through AP3B2-dependent trafficking, linking the adaptor to cytoskeletal-coupled cell behavior and neuronal architecture.","evidence":"4.1G KO MEFs and mice, in vitro binding, surface FACS, migration and FAK assays, retinal histology, optokinetic testing, neurite assays","pmids":["26644476","25660028"],"confidence":"High","gaps":["Whether 4.1G stabilizes or traffics surface β1 integrin not distinguished","AP3B2-dependent trafficking step molecularly undefined"]},{"year":2017,"claim":"Connected 4.1G's Schwann-cell scaffolding role to functional myelin physiology—loss disrupts Lin7 sorting (via MPP6) and produces myelin and nerve-conduction defects.","evidence":"4.1G KO mice, EM, electrophysiology, Co-IP (MPP6-Lin7), immunofluorescence","pmids":["28755316"],"confidence":"High","gaps":["Causal chain from Lin7 mislocalization to conduction defect incomplete","Cell-autonomy not fully dissected"]},{"year":2019,"claim":"Pinpointed the direct molecular basis for cAMP suppression: 4.1G-FERM binds a triple-arginine motif in the AC6 N-terminus to control AC6 membrane distribution and inhibit its catalytic activity, attenuating PTHR-Gs signaling.","evidence":"In vitro binding, AC6-N-3A mutagenesis, competitive inhibition, siRNA, cAMP assays, Co-IP","pmids":["31383768"],"confidence":"High","gaps":["Structural detail of the FERM-AC6N interface not solved","Whether other AC isoforms are similarly regulated unknown"]},{"year":2022,"claim":"Identified a developmental signaling role: 4.1G is required for primary ciliogenesis and cilia-dependent Hedgehog signaling driving osteoblast differentiation and bone mineralization.","evidence":"4.1G KO mice, MC3T3-E1 knockdown, ciliary imaging, calcium-deposition and Hedgehog assays","pmids":["35216233"],"confidence":"Medium","gaps":["Molecular mechanism linking 4.1G to ciliary assembly not defined","Direct ciliary partner not identified"]},{"year":2023,"claim":"Generalized 4.1G's GPCR-scaffolding role to orphan SREB receptors, showing it promotes SREB1 plasma-membrane localization and alters its membrane microenvironment.","evidence":"BioID2 proximity labeling, MS, Co-IP, siRNA knockdown, immunofluorescence","pmids":["37998360"],"confidence":"Medium","gaps":["Direct vs. proximity interaction not fully separated","Functional consequence for SREB signaling untested"]},{"year":2025,"claim":"Characterized the CTD as intrinsically disordered, forming a fuzzy, crowding-enhanced complex with NuMA—extending 4.1G's interaction repertoire to a disorder-driven binding mode sensitive to the cellular environment.","evidence":"SAXS, NMR, in vitro binding kinetics under macromolecular crowding","pmids":["40726410"],"confidence":"High","gaps":["Cellular role of the 4.1G-NuMA interaction not established","Whether crowding-dependent binding occurs in vivo untested"]},{"year":null,"claim":"It remains unknown how the distinct partner repertoires (GPCRs, integrins, adenylyl cyclases, scaffolds, NuMA) are coordinated by a single adaptor across tissues, and whether EPB41L2 dysfunction underlies any human disease.","evidence":"","pmids":[],"confidence":"Low","gaps":["No human disease link established in the corpus","No integrated structural model of full-length 4.1G with multiple partners","Tissue-specific partner selection mechanism undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,3,4,6,20,23,25]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[15,23,2,3]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,12,20]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[11,14,22]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[15,20,23,25,4]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,0]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[12]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[24]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3,4,15,23,24]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[20]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[4,20,25,21]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[11,14,22,21]}],"complexes":[],"partners":["MPP6","NECL4","ITGB1","ADCY6","FCGR1A","AP3B2","NUMA1","CADM1"],"other_free_text":[]}},"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 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Model.","date":"2020","source":"Nutrients","url":"https://pubmed.ncbi.nlm.nih.gov/32235804","citation_count":7,"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":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":"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 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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":18326,"output_tokens":6850,"usd":0.078864,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15955,"output_tokens":5464,"usd":0.108188,"stage2_stop_reason":"end_turn"},"total_usd":0.187052,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","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 regions of high homology to erythrocyte protein 4.1R: a membrane-binding domain, a spectrin-actin binding domain, and a C-terminal domain. Specific isoforms of 4.1G exhibit differential subcellular localizations, resulting from both alternative splicing and distinct gene expression.\",\n      \"method\": \"cDNA cloning, sequence analysis, subcellular localization studies\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cDNA cloning with sequence analysis and subcellular localization, single lab but multiple methods\",\n      \"pmids\": [\"9598318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"4.1G binds the carboxyl-terminal domain of the T cell adhesion molecule PTA-1 (CD226) and also associates with human discs large (hDlg). T cell stimulation causes PTA-1 and 4.1G to associate tightly with the cytoskeleton, and activated cells show altered binding of PTA-1 to the amino-terminal region of 4.1G, forming a dynamic molecular complex.\",\n      \"method\": \"Co-immunoprecipitation, membrane raft fractionation, cytoskeletal association assays, domain-binding studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with domain-mapping, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"15138281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"4.1G binds to the third intracellular loop of the A1 adenosine receptor (A1AR) via its C-terminal domain. This interaction was confirmed in brain tissue and in HEK-293 and CHO cells. 4.1G overexpression reduced A1AR-mediated inhibition of cAMP accumulation, intracellular calcium release, and altered cell-surface A1AR expression.\",\n      \"method\": \"Yeast two-hybrid screening, truncation binding studies, co-immunoprecipitation in 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 / Strong — yeast two-hybrid identification, confirmed by Co-IP in native tissue, functional readouts with multiple cell lines, multiple orthogonal methods\",\n      \"pmids\": [\"12974671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"4.1G directly interacts with the metabotropic glutamate receptor subtype 1alpha (mGlu1alpha) via the C-terminal tail of mGlu1alpha, co-localizes with mGlu1alpha in hippocampal neurons, and modulates mGlu1alpha-mediated cAMP accumulation, ligand-binding ability, and cellular distribution.\",\n      \"method\": \"Co-localization in hippocampal neurons, co-immunoprecipitation in HEK-293 cells and rat brain tissue, domain truncation analysis, functional cAMP assays\",\n      \"journal\": \"Journal of neuroscience research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP in native brain tissue and transfected cells, co-localization in neurons, functional assays, multiple orthogonal methods\",\n      \"pmids\": [\"15372499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"4.1G interacts with the C-terminus of the parathyroid hormone receptor (PTHR) and facilitates cell-surface localization of PTHR, as shown by cell-surface biotinylation. The full-length 4.1G (but not 4.1G-CTD dominant-negative) enhanced PTH-stimulated ERK1/2 phosphorylation and intracellular Ca2+ elevation.\",\n      \"method\": \"Yeast two-hybrid, co-localization in COS-7 cells, cell-surface biotinylation assay, ERK phosphorylation and Ca2+ assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid, cell-surface biotinylation with dominant-negative controls, functional signaling assays, multiple orthogonal methods\",\n      \"pmids\": [\"16029167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"4.1G is expressed in Schwann cells of the peripheral nervous system and is specifically localized at paranodal loops, Schmidt-Lanterman incisures, and periaxonal, mesaxonal, and abaxonal membranes. During development, 4.1G transitions from diffuse distribution in immature Schwann cells to discrete localization at these membrane specializations during myelination.\",\n      \"method\": \"Northern blot, Western blot, immunohistochemistry with specific antibody, double immunolabeling, immunoelectron microscopy\",\n      \"journal\": \"Journal of neuroscience research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple immunolabeling methods including electron microscopy, single lab\",\n      \"pmids\": [\"16752423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The C-terminal domain of 4.1G interacts with the cytoplasmic tail of FcγRI (CD64). A specific Fc gamma RI membrane-proximal core motif of HxxBxxxBB followed by hydrophobic and charged residues is central for 4.1G interaction, identified by Fc gamma RI truncation and alanine-substitution mutant analysis.\",\n      \"method\": \"Yeast two-hybrid, domain truncation analysis, alanine substitution mutagenesis\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid with systematic mutagenesis, single lab\",\n      \"pmids\": [\"18023480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Mice with deletion of 4.1G and knockdown of 4.1N to ~22% of wild-type levels (combined ~12% hippocampal expression) showed a moderate reduction in synaptosomal GluR1 at 3 weeks of age, but no change in basic glutamatergic synaptic transmission or long-term potentiation, indicating 4.1G and 4.1N do not have a crucial role in glutamatergic synaptic transmission.\",\n      \"method\": \"Knockout and knockdown mouse model, electrophysiology, synaptosomal fractionation and immunoblotting\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO combined with electrophysiology and biochemical fractionation; this is a negative result experimentally established with rigorous controls\",\n      \"pmids\": [\"19225127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"4.1G associates with cell adhesion molecule-1 (CADM1) in seminiferous tubule lysates, as shown by co-immunoprecipitation. 4.1G is immunolocalized along cell membranes of Sertoli cells, spermatogonia, and early spermatocytes, and is expressed in spermatogonial stem cells at cell-cell contact regions.\",\n      \"method\": \"Immunolocalization, immunoprecipitation, in vitro spermatogonial stem cell culture, immunoblotting\",\n      \"journal\": \"Reproduction (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP in native tissue, confirmed localization by multiple methods, single lab\",\n      \"pmids\": [\"20200204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"4.1G binds erythroid membrane proteins including band 3, glycophorin C, CD44, and p55 via its membrane-binding domain. The N-terminal headpiece region of 4.1G differentiates its binding affinities from those of 4.1R135 for band 3 and glycophorin C. The headpiece also contains a high-affinity calcium-dependent calmodulin-binding site that modulates interactions with these membrane proteins.\",\n      \"method\": \"In vitro binding assays, affinity characterization, calmodulin interaction studies\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstituted binding assays with defined domain constructs and calmodulin regulation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"20812914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"4.1G deficiency in mice (B6-129 hybrid background) causes male infertility associated with atrophy, impaired cell-cell contact, and sloughing of spermatogenic cells. 4.1G associates with NECL4 (nectin-like 4) in Sertoli cells, and NECL4 expression is decreased and mislocalized in 4.1G-/- testis.\",\n      \"method\": \"Knockout mouse model, histology, ultrastructural analysis (electron microscopy), co-immunoprecipitation, immunolocalization\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with defined infertility phenotype, EM ultrastructure, Co-IP, and immunolocalization, multiple orthogonal methods\",\n      \"pmids\": [\"21482674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"4.1G co-localizes with MPP6 at Schmidt-Lanterman incisures and paranodes in sciatic nerve. MPP6 co-immunoprecipitates with 4.1G. In 4.1G knockout mice, MPP6 is mislocalized to the cytoplasm near Schwann cell nuclei, demonstrating that 4.1G is required for targeting MPP6 to Schmidt-Lanterman incisures.\",\n      \"method\": \"Immunofluorescence co-localization, co-immunoprecipitation, 4.1G knockout mouse analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP in native tissue confirmed with KO mouse showing specific mislocalization, multiple orthogonal methods\",\n      \"pmids\": [\"22025680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In heart muscle cells, 4.1G is localized to intracellular structures coincident with sarcoplasmic reticulum and exists in an immunoprecipitable complex with spectrin and SERCA2.\",\n      \"method\": \"Immunofluorescence, immunoprecipitation, subcellular fractionation\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and subcellular fractionation showing complex with SERCA2, single lab\",\n      \"pmids\": [\"22429617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Serine phosphorylation of the FcγRI cytoplasmic tail by CK2 promotes preferential interaction with protein 4.1G in vitro. 4.1G co-localizes with FcγRI in unstimulated U937 cells where CY is constitutively serine-phosphorylated; FcγRI cross-linking causes uncoupling. A nonphosphorylatable FcγRI mutant is excluded from lipid rafts, implicating 4.1G in phosphoserine-dependent targeting of FcγRI to lipid rafts.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding with CK2-phosphorylated peptides, co-immunoprecipitation in human PBMC, immunostaining, lipid raft fractionation, mutagenesis\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro phosphorylation-dependent binding with mutagenesis, confirmed by Co-IP in primary cells and lipid raft fractionation, multiple orthogonal methods\",\n      \"pmids\": [\"22003208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Deletion of 4.1G in Schwann cells causes aberrant distribution of internodal proteins including juxtaparanodal Kv1 channels, Caspr2, and TAG-1, and paranodal junction components. In 4.1G-/- mice, these proteins aggregate at the juxtaparanodal region rather than forming the normal double strand flanking paranodal junction components along internodes.\",\n      \"method\": \"4.1G knockout mouse, immunofluorescence, confocal microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with defined molecular phenotype in axoglial organization, replicated across multiple protein markers\",\n      \"pmids\": [\"22291039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"4.1G overexpression suppresses forskolin-induced and PTH-stimulated cAMP production in HEK293 cells; 4.1G knockdown increases cAMP production. A FERM-domain-deleted 4.1G mutant lacking plasma membrane distribution does not alter cAMP production, indicating that plasma membrane association of 4.1G is required for its suppression of adenylyl cyclase activity.\",\n      \"method\": \"Overexpression and siRNA knockdown in HEK293 cells, cAMP assay, membrane fractionation, FERM deletion mutagenesis\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — gain- and loss-of-function with domain mutagenesis and membrane fractionation, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"23201780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"4.1G overexpression promotes arborization of oligodendrocyte cell line OLN-93 through its FERM domain, while FERM-domain-deleted 4.1G does not. 4.1G also promotes tight junction reassembly (shown by calcium switch experiment) and its knockdown inhibits tight junction formation, with 4.1G co-clustering with ZO-1 at cell periphery.\",\n      \"method\": \"Overexpression and siRNA knockdown in OLN-93 cells, calcium switch assay, immunoprecipitation, immunofluorescence, domain deletion analysis\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with domain mutagenesis and 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 channels in rod outer segments (ROS) through its FERM and CTD domains, identified by immunoprecipitation/mass spectrometry and confirmed by truncation and domain-binding assays. A smaller splice variant of 4.1G selectively interacted with CNG channels not associated with the peripherin-2-CNG channel complex.\",\n      \"method\": \"Immunoprecipitation and mass spectrometry, domain truncation analysis, domain-binding assays, immunofluorescence\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — IP-MS identification followed by systematic domain truncation and binding assays, multiple orthogonal methods\",\n      \"pmids\": [\"24144699\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Src kinase is present in Schmidt-Lanterman incisures and forms a complex with MPP6. In 4.1G-deficient nerve fibers (which lack both 4.1G and MPP6 from SLIs), active (P418) Src immunoreactivity in SLIs is enhanced compared to wild-type, implicating the 4.1G-MPP6 complex in restraining Src activity at SLIs.\",\n      \"method\": \"Immunostaining with phospho-specific antibodies, 4.1G knockout mouse analysis, immunoprecipitation\",\n      \"journal\": \"Histochemistry and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of Src-MPP6 plus KO phenotype analysis, single lab\",\n      \"pmids\": [\"23306908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Ca2+/calmodulin binds to the N-terminal headpiece region (GHP) of 4.1G at the peptide SRGISRFIPPWLKKQKS, inducing a conformational switch from intrinsically disordered coiled structure to compact structure. This structural change sterically inhibits 4.1G FERM domain interactions with membrane proteins.\",\n      \"method\": \"Small-angle X-ray scattering, NMR spectroscopy, circular dichroism spectroscopy, peptide binding assays\",\n      \"journal\": \"Cell biochemistry and biophysics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — three orthogonal structural biophysical methods identifying the Ca2+/CaM binding site and conformational mechanism, single lab\",\n      \"pmids\": [\"23354586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"4.1G binds directly to β1 integrin via its membrane-binding domain (shown by Co-IP and in vitro binding assays). In 4.1G-/- mouse embryonic fibroblasts, cell surface expression of β1 integrin and its active form are decreased, adhesion, spreading, and migration are impaired, and focal adhesion kinase phosphorylation is suppressed.\",\n      \"method\": \"4.1G knockout MEF cells, co-immunoprecipitation, in vitro binding assay, cell-surface FACS, migration assays, FAK phosphorylation analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct in vitro binding assay confirmed with KO cells showing surface integrin reduction, downstream signaling changes, and functional phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"26644476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"4.1G is highly expressed in retinal photoreceptors and binds to AP3B2 (a protein involved in neuronal membrane trafficking). 4.1G-deficient retinas show mislocalization of photoreceptor terminals (without loss of synaptic connections), and 4.1G promotes neurite extension in an AP3B2-dependent manner. 4.1G mutant mice show visual acuity impairment.\",\n      \"method\": \"4.1G KO mouse, protein interaction (binding assay), immunohistochemistry, optokinetic response test, neurite extension assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with defined anatomical phenotype, binding partner identification, functional rescue/dependency demonstrated with AP3B2, multiple orthogonal methods\",\n      \"pmids\": [\"25660028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"4.1G deficiency in mice causes myelin abnormalities in the peripheral nervous system (thicker myelin internodes, distorted paranodal tips), slowed motor-conduction velocity, and loss of Lin7c and Lin7a (scaffold proteins) from sciatic nerves. MPP6 interacts with Lin7 by immunoprecipitation, and 4.1G is required for proper Lin7 sorting in Schwann cells.\",\n      \"method\": \"4.1G-/- mouse model, electron microscopy, electrophysiology, immunoprecipitation, immunofluorescence\",\n      \"journal\": \"Histochemistry and cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with EM, electrophysiology, Co-IP showing MPP6-Lin7 interaction, multiple orthogonal methods\",\n      \"pmids\": [\"28755316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"4.1G directly and selectively binds to the N-terminus of adenylyl cyclase type 6 (AC6) via its FERM domain, as shown by in vitro binding assays. Three consecutive arginine residues in AC6-N are required for 4.1G-FERM binding and for proper plasma membrane distribution of AC6. This interaction suppresses AC6 catalytic activity, attenuating PTHR-mediated Gs/AC6/cAMP signaling.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assay, site-directed mutagenesis (AC6-N-3A), competitive inhibition with AC6-N overexpression, siRNA knockdown, cAMP assay\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro direct binding with mutagenesis, confirmed by Co-IP in cells, competitive inhibitor approach and knockdown, multiple rigorous methods\",\n      \"pmids\": [\"31383768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"4.1G is expressed in bone and is required for primary ciliogenesis and osteoblast differentiation. In 4.1G-knockout mice, calcium deposits and primary cilium formation are suppressed in preosteoblast-rich trabecular bone. Knockdown of 4.1G in MC3T3-E1 cells suppresses cilium elongation and inhibits cilia-mediated Hedgehog signaling and subsequent osteoblast differentiation.\",\n      \"method\": \"4.1G KO mouse, siRNA knockdown in MC3T3-E1 cells, immunofluorescence, calcium deposition assay, Hedgehog signaling assay\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse phenotype confirmed by cell-based knockdown with defined molecular pathway (Hedgehog), single lab\",\n      \"pmids\": [\"35216233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EPB41L2 (4.1G) is a proximity interactor of Super-Conserved Receptors Expressed in the Brain (SREBs), confirmed by BioID2 proximity labeling and co-immunoprecipitation. EPB41L2 promotes plasma membrane localization of SREB1 and modifies SREB1 membrane microenvironment (increased detergent solubilization) as shown by siRNA knockdown.\",\n      \"method\": \"BioID2 proximity labeling, mass spectrometry, co-immunoprecipitation, siRNA knockdown, immunofluorescence\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity labeling with MS confirmed by Co-IP, 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 disorder, enhances binding affinity for NuMA, and accelerates association kinetics.\",\n      \"method\": \"Small-angle X-ray scattering, NMR spectroscopy, biophysical binding assays under crowding conditions\",\n      \"journal\": \"Physical chemistry chemical physics : PCCP\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple structural biophysical methods (SAXS, NMR) with binding kinetics analysis; interaction with NuMA established in vitro\",\n      \"pmids\": [\"40726410\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EPB41L2 (4.1G) is a multidomain membrane skeletal adaptor protein whose FERM domain anchors it to the plasma membrane and mediates direct binding to diverse transmembrane proteins—including GPCRs (A1AR, mGlu1α, PTHR, SREB1), integrins (β1), ion channels (CNG channels, adenylyl cyclase AC6), and immune receptors (FcγRI, PTA-1/CD226)—while its CTD interacts with NuMA and scaffolding proteins (hDlg, MPP6, Lin7); through these interactions 4.1G regulates cell-surface localization and signaling of its binding partners (promoting receptor surface expression, suppressing AC6/cAMP output via direct FERM-AC6N binding), organizes internodal and Schmidt-Lanterman incisure architecture in Schwann cells by targeting MPP6 and restraining Src activity, supports spermatogenic cell adhesion via NECL4, drives photoreceptor synapse positioning via AP3B2-dependent membrane trafficking, facilitates cell adhesion and migration by maintaining surface-active β1 integrin, and promotes primary ciliogenesis and Hedgehog-mediated osteoblast differentiation; calmodulin binding to the N-terminal headpiece induces a disorder-to-order transition that allosterically inhibits FERM domain interactions, providing Ca2+-dependent regulation of the protein's scaffolding activity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EPB41L2 (4.1G) is a multidomain membrane-skeletal adaptor that organizes the cell surface by linking transmembrane proteins to the underlying cytoskeleton, built from a membrane-binding (FERM) domain, a spectrin-actin binding domain, and a C-terminal domain (CTD) [#0]. Through its FERM domain it docks directly to diverse membrane partners—\\u03b21 integrin [#20] and the N-terminus of adenylyl cyclase type 6 (AC6) via a triple-arginine motif [#23]—while its CTD engages G-protein-coupled receptors including the A1 adenosine receptor [#2], mGlu1\\u03b1 [#3], and the parathyroid hormone receptor [#4], as well as the immune receptor Fc\\u03b3RI [#6] and CNG channels of photoreceptors [#17]. By controlling the cell-surface delivery and microenvironment of these partners, 4.1G tunes their signaling output: it promotes surface expression of PTHR and \\u03b21 integrin [#4, #20] yet directly suppresses AC6 catalytic activity to dampen Gs/cAMP signaling, an effect requiring FERM-dependent plasma-membrane association [#15, #23]. In Schwann cells 4.1G localizes to paranodes and Schmidt-Lanterman incisures and is required to target the scaffold MPP6 and Lin7 proteins, organize internodal channel distribution, and restrain Src activity, with its loss causing myelin abnormalities and slowed nerve conduction [#11, #14, #18, #22]. 4.1G also supports spermatogenic cell adhesion through NECL4 (loss causes male infertility) [#10, #11], positions photoreceptor synaptic terminals via AP3B2-dependent trafficking [#21], and promotes primary ciliogenesis and Hedgehog-driven osteoblast differentiation [#24]. Its scaffolding is allosterically gated: Ca2+/calmodulin binding to the N-terminal headpiece drives a disorder-to-order transition that sterically inhibits FERM-domain interactions, providing Ca2+-dependent regulation [#9, #19], while the intrinsically disordered CTD forms a fuzzy, crowding-sensitive complex with NuMA [#26].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established 4.1G as a protein 4.1R paralog with a conserved tripartite domain architecture, framing it as a candidate membrane-skeletal adaptor with isoform-specific localization.\",\n      \"evidence\": \"cDNA cloning, sequence analysis, and subcellular localization of isoforms\",\n      \"pmids\": [\"9598318\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No binding partners or function identified\", \"Isoform-specific roles not assigned\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrated that 4.1G acts as a receptor-anchoring scaffold by binding GPCR intracellular domains (A1AR, mGlu1\\u03b1) and the adhesion molecule PTA-1/CD226, modulating receptor surface expression and downstream cAMP/Ca2+ signaling.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP in brain tissue and transfected cells, functional cAMP/Ca2+ assays\",\n      \"pmids\": [\"15138281\", \"12974671\", \"15372499\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Domain responsible for receptor binding not fully delineated across partners\", \"Whether 4.1G acts in receptor trafficking vs. retention unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed 4.1G facilitates cell-surface localization of a GPCR (PTHR) and amplifies its downstream signaling, requiring full-length protein, establishing a positive trafficking/scaffolding role distinct from signal suppression.\",\n      \"evidence\": \"Yeast two-hybrid, cell-surface biotinylation with CTD dominant-negative, ERK and Ca2+ assays in COS-7\",\n      \"pmids\": [\"16029167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of surface delivery (trafficking vs. stabilization) not resolved\", \"Not validated in native tissue\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Mapped a defined membrane-proximal motif (HxxBxxxBB) in Fc\\u03b3RI required for CTD binding, providing a sequence basis for 4.1G recognition of immune-receptor tails.\",\n      \"evidence\": \"Yeast two-hybrid, truncation and alanine-scanning mutagenesis\",\n      \"pmids\": [\"18023480\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interaction not confirmed in primary immune cells at this stage\", \"Functional consequence not yet shown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Genetic loss-of-function established that 4.1G (with 4.1N) is not essential for glutamatergic synaptic transmission or LTP, bounding its in vivo role in the hippocampus despite GluR1 changes.\",\n      \"evidence\": \"4.1G KO / 4.1N knockdown mice, electrophysiology, synaptosomal immunoblotting\",\n      \"pmids\": [\"19225127\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Redundancy with other 4.1 family members not fully excluded\", \"Subtle synaptic phenotypes not probed\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined 4.1G's membrane-binding domain interactions with classical erythroid skeletal partners and adhesion molecules, and identified a Ca2+-dependent calmodulin site in the headpiece that modulates these interactions—first hint of allosteric regulation.\",\n      \"evidence\": \"In vitro binding/affinity assays, calmodulin interaction studies; Co-IP and immunolocalization in seminiferous tubules (CADM1)\",\n      \"pmids\": [\"20812914\", \"20200204\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CaM regulation not yet defined\", \"Physiological context of membrane-protein binding incomplete\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"In vivo KO models established 4.1G as essential for organizing membrane specializations: it targets MPP6 to Schmidt-Lanterman incisures in Schwann cells and supports spermatogenic adhesion via NECL4, with loss causing male infertility.\",\n      \"evidence\": \"4.1G KO mice, EM ultrastructure, Co-IP, immunolocalization in testis and sciatic nerve; SERCA2/spectrin complex in heart\",\n      \"pmids\": [\"21482674\", \"22025680\", \"22429617\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking MPP6 mislocalization to myelin defects not yet defined\", \"SERCA2 complex functional role uncharacterized\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved the mechanistic basis of 4.1G-mediated signal suppression and membrane organization—FERM-dependent plasma-membrane association is required to suppress adenylyl cyclase/cAMP output and to organize internodal channels (Kv1, Caspr2, TAG-1) and tight junctions.\",\n      \"evidence\": \"Gain/loss of function with FERM-deletion mutants, cAMP assays, KO mouse internodal marker imaging, calcium-switch tight-junction assays\",\n      \"pmids\": [\"23201780\", \"22291039\", \"21898413\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The adenylyl cyclase isoform directly bound not yet identified at this stage\", \"Link between membrane scaffolding and channel clustering mechanism unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended the partner repertoire to photoreceptor CNG channels and demonstrated phosphoserine-dependent partner selection (CK2-phosphorylated Fc\\u03b3RI) and a role in restraining Src at incisures, revealing post-translational and signaling control of 4.1G scaffolding.\",\n      \"evidence\": \"IP-MS, domain-binding assays, in vitro CK2-phosphopeptide binding, Co-IP in primary cells, lipid-raft fractionation, KO Src phospho-imaging\",\n      \"pmids\": [\"24144699\", \"22003208\", \"23306908\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How phosphorylation switches partner affinity structurally not resolved\", \"Mechanism by which 4.1G-MPP6 restrains Src unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Provided the structural mechanism for Ca2+ regulation: calmodulin binding to a defined headpiece peptide drives a disorder-to-order transition that sterically inhibits FERM-domain membrane interactions.\",\n      \"evidence\": \"SAXS, NMR, circular dichroism, peptide binding assays\",\n      \"pmids\": [\"23354586\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulation not demonstrated for full-length protein in cells\", \"Which membrane partners are most sensitive to CaM gating untested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"KO-based studies established 4.1G as a determinant of \\u03b21-integrin surface availability and adhesion/migration, and of photoreceptor terminal positioning through AP3B2-dependent trafficking, linking the adaptor to cytoskeletal-coupled cell behavior and neuronal architecture.\",\n      \"evidence\": \"4.1G KO MEFs and mice, in vitro binding, surface FACS, migration and FAK assays, retinal histology, optokinetic testing, neurite assays\",\n      \"pmids\": [\"26644476\", \"25660028\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether 4.1G stabilizes or traffics surface \\u03b21 integrin not distinguished\", \"AP3B2-dependent trafficking step molecularly undefined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected 4.1G's Schwann-cell scaffolding role to functional myelin physiology—loss disrupts Lin7 sorting (via MPP6) and produces myelin and nerve-conduction defects.\",\n      \"evidence\": \"4.1G KO mice, EM, electrophysiology, Co-IP (MPP6-Lin7), immunofluorescence\",\n      \"pmids\": [\"28755316\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Causal chain from Lin7 mislocalization to conduction defect incomplete\", \"Cell-autonomy not fully dissected\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Pinpointed the direct molecular basis for cAMP suppression: 4.1G-FERM binds a triple-arginine motif in the AC6 N-terminus to control AC6 membrane distribution and inhibit its catalytic activity, attenuating PTHR-Gs signaling.\",\n      \"evidence\": \"In vitro binding, AC6-N-3A mutagenesis, competitive inhibition, siRNA, cAMP assays, Co-IP\",\n      \"pmids\": [\"31383768\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural detail of the FERM-AC6N interface not solved\", \"Whether other AC isoforms are similarly regulated unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified a developmental signaling role: 4.1G is required for primary ciliogenesis and cilia-dependent Hedgehog signaling driving osteoblast differentiation and bone mineralization.\",\n      \"evidence\": \"4.1G KO mice, MC3T3-E1 knockdown, ciliary imaging, calcium-deposition and Hedgehog assays\",\n      \"pmids\": [\"35216233\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism linking 4.1G to ciliary assembly not defined\", \"Direct ciliary partner not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Generalized 4.1G's GPCR-scaffolding role to orphan SREB receptors, showing it promotes SREB1 plasma-membrane localization and alters its membrane microenvironment.\",\n      \"evidence\": \"BioID2 proximity labeling, MS, Co-IP, siRNA knockdown, immunofluorescence\",\n      \"pmids\": [\"37998360\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. proximity interaction not fully separated\", \"Functional consequence for SREB signaling untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Characterized the CTD as intrinsically disordered, forming a fuzzy, crowding-enhanced complex with NuMA—extending 4.1G's interaction repertoire to a disorder-driven binding mode sensitive to the cellular environment.\",\n      \"evidence\": \"SAXS, NMR, in vitro binding kinetics under macromolecular crowding\",\n      \"pmids\": [\"40726410\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular role of the 4.1G-NuMA interaction not established\", \"Whether crowding-dependent binding occurs in vivo untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how the distinct partner repertoires (GPCRs, integrins, adenylyl cyclases, scaffolds, NuMA) are coordinated by a single adaptor across tissues, and whether EPB41L2 dysfunction underlies any human disease.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No human disease link established in the corpus\", \"No integrated structural model of full-length 4.1G with multiple partners\", \"Tissue-specific partner selection mechanism undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 3, 4, 6, 20, 23, 25]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [15, 23, 2, 3]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 12, 20]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [11, 14, 22]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [15, 20, 23, 25, 4]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 0]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 4, 15, 23, 24]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [20]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [4, 20, 25, 21]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [11, 14, 22, 21]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MPP6\", \"NECL4\", \"ITGB1\", \"ADCY6\", \"FCGR1A\", \"AP3B2\", \"NUMA1\", \"CADM1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":5,"faith_total":5,"faith_pct":100.0}}