{"gene":"VSNL1","run_date":"2026-04-28T23:00:23","timeline":{"discoveries":[{"year":1992,"finding":"VILIP (VSNL1 ortholog in chicken) was identified as a neuronal EF-hand Ca2+-binding protein with four EF-hand motifs, showing 40-46% sequence identity to retinal calcium-binding proteins visinin and recoverin, and expressed widely in brain but not in liver, heart, or skeletal muscle.","method":"cDNA cloning, Northern analysis, in situ hybridization","journal":"Brain research. Molecular brain research","confidence":"High","confidence_rationale":"Tier 1 — original cloning and structural characterization with multiple methods","pmids":["1359372"],"is_preprint":false},{"year":1994,"finding":"VILIP (VSNL1) binds only two active Ca2+/Mg2+ sites (not four), with Ca2+ affinity K'Ca of 1.0×10^6 M-1 and Mg2+ affinity K'Mg of 4.8×10^3 M-1; Ca2+ binding causes 20-30% increase in Trp fluorescence indicating conformational change; Mg2+ form resembles metal-free form unlike NCS-1.","method":"Flow dialysis (Ca2+ binding), equilibrium gel filtration (Mg2+ binding), Trp fluorescence spectroscopy, near UV difference spectra, DTNB thiol reactivity assay on recombinant proteins expressed in E. coli","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — multiple orthogonal in vitro biophysical assays on recombinant protein","pmids":["7806504"],"is_preprint":false},{"year":1996,"finding":"VILIP (VSNL1) exists in cytoplasmic, membrane-associated, and cytoskeleton-associated pools; its association with the cytoskeletal fraction is Ca2+-dependent and mediated by direct interaction with actin, as Ca2+-loaded recombinant VILIP binds actin in overlay assay, β-actin co-immunoprecipitates with native VILIP from brain extracts in presence of Ca2+, and actin and VILIP co-localize in VILIP-transfected PC12 cells.","method":"Cell fractionation, actin overlay assay, co-immunoprecipitation, immunocytochemistry in transfected PC12 cells","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods including in vitro binding, co-IP, and co-localization","pmids":["8780737"],"is_preprint":false},{"year":1997,"finding":"VILIP-1 (VSNL1) increases β-adrenergic receptor-stimulated cAMP levels in C6 glioma cells, acting downstream of receptor and G protein (also stimulated by forskolin); this effect requires N-terminal myristoylation, as the myristoylation-deficient mutant reduces cAMP levels instead; myristoylated wild-type VILIP associates with membranes in a Ca2+-dependent manner (calcium-myristoyl switch) while the non-myristoylated mutant shows strongly reduced membrane association.","method":"Stable transfection in C6 glioma cells, cAMP assay, subcellular fractionation, site-directed mutagenesis of myristoylation site","journal":"Journal of neurochemistry","confidence":"High","confidence_rationale":"Tier 1-2 — functional assay with mutagenesis and subcellular fractionation, defining mechanism","pmids":["9109541"],"is_preprint":false},{"year":1997,"finding":"VILIP (VSNL1) is expressed in olfactory sensory cell cilia and dendritic knobs, and recombinant VILIP attenuates odorant-induced cAMP formation in olfactory cilia preparations in a Ca2+-dependent manner, likely by directly affecting adenylyl cyclase (also inhibits forskolin-induced cAMP formation), without interfering with receptor desensitization.","method":"Immunolocalization, in vitro cAMP assay with olfactory cilia preparations using recombinant VILIP","journal":"European journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro functional assay with recombinant protein, single lab","pmids":["9157011"],"is_preprint":false},{"year":1997,"finding":"Low-level expression of myristoylated wild-type VILIP in C6 cells elevates basal cAMP and induces astrocyte-like differentiation with increased GFAP expression; non-myristoylated VILIP mutant reduces cAMP and GFAP without inducing differentiated morphology, demonstrating myristoylation-dependent cAMP regulation drives differentiation.","method":"Stable transfection in C6 cells, cAMP measurement, immunostaining for GFAP, morphological analysis","journal":"Neuroscience letters","confidence":"Medium","confidence_rationale":"Tier 2 — functional assay with mutagenesis, single lab","pmids":["9364517"],"is_preprint":false},{"year":1997,"finding":"Wild-type VILIP in PC12 cells associates strongly with cell membranes (especially cell-cell contacts) in a Ca2+-dependent manner, whereas myristoylation-deficient VILIP distributes more evenly with much less membrane association, demonstrating the calcium-myristoyl switch as the primary but not sole determinant of membrane targeting.","method":"Stable transfection in PC12 cells, immunocytochemistry, subcellular fractionation and Western blot","journal":"Neuroscience letters","confidence":"Medium","confidence_rationale":"Tier 2 — localization with functional mutagenesis, single lab","pmids":["9147390"],"is_preprint":false},{"year":1999,"finding":"VILIP (VSNL1) contains a double-stranded RNA-binding domain and specifically binds dsRNA in a Ca2+-dependent manner as a single protein-RNA complex (Kd ~9×10^-6 M); VILIP specifically binds the 3'-UTR of the trkB neurotrophin receptor mRNA, suggesting a role in Ca2+-dependent regulation of localized mRNA in hippocampal dendrites.","method":"Mobility shift assay, RNA binding assay with recombinant VILIP, domain analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro binding assay with defined Kd and specificity, single lab","pmids":["10531361"],"is_preprint":false},{"year":2001,"finding":"VILIP-1 (VSNL1) expression in PC12 cells enhances ionomycin-induced cytotoxicity and increases tau hyperphosphorylation compared to control or calbindin-D28K-transfected cells; co-expression of calbindin-D28K rescues VILIP-1-mediated cytotoxicity; VILIP-1 associates with amyloid plaques and extracellular tangles in Alzheimer's disease brains.","method":"Stable transfection of PC12 cells, cytotoxicity assays, tau phosphorylation immunoblot, immunohistochemistry in AD brain tissue","journal":"Neurobiology of disease","confidence":"Medium","confidence_rationale":"Tier 2 — cell-based functional assay with transfection and defined molecular readouts, single lab","pmids":["11592857"],"is_preprint":false},{"year":2001,"finding":"VILIP-1 (VSNL1) increases cGMP levels in transfected C6 and PC12 cells; membrane-associated (myristoylated) VILIP-1 preferentially stimulates particulate guanylyl cyclase (GC), while cytosolic (non-myristoylated) VILIP-1 enhances soluble GC; VILIP-1 physically interacts with the catalytic domain of particulate GC-A and GC-B and with native GC enriched from rat brain, demonstrated by GST pull-down and surface plasmon resonance; VILIP-1 introduced into cerebellar granule cells specifically influences GC-B but not GC-A signaling.","method":"Transfection in C6/PC12 cells, cGMP assay, GST pull-down, surface plasmon resonance, trituration of recombinant protein into cerebellar granule cells","journal":"Journal of neurochemistry","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods including direct binding (SPR + pull-down) and functional assays with subcellular localization control","pmids":["11579136"],"is_preprint":false},{"year":2002,"finding":"VILIP-1 (VSNL1) undergoes a reversible, stimulus-dependent calcium-myristoyl switch in living hippocampal neurons: GFP-tagged VILIP-1 translocates from diffuse cytosolic distribution to plasma membrane and Golgi membranes upon Ca2+ ionophore treatment or depolarization/glutamate receptor activation; this translocation is fully reversed by EGTA; myristoylation-deficient VILIP-1-GFP does not translocate.","method":"Live fluorescence microscopy of GFP-tagged VILIP-1 in transfected NG108-15, COS-7 cells and primary hippocampal neurons; Ca2+ ionophore and EGTA manipulation; myristoylation-deficient mutant","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — direct live-cell imaging with reversibility controls and mutagenesis, replicated in multiple cell types","pmids":["12196554"],"is_preprint":false},{"year":2002,"finding":"VILIP-1 and VILIP-3 show distinct Ca2+-dependent subcellular localizations and activate different cellular signaling pathways; VILIP-3 (mainly in Purkinje cells) and VILIP-1 (in granule cells) have different protein interaction partners and different degrees of Ca2+-dependent membrane association, indicating cell-type-specific signaling functions.","method":"Subcellular fractionation, cGMP signaling assays, protein interaction assays in transfected cells, immunolocalization","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 — comparative functional and localization analysis, single lab","pmids":["12445467"],"is_preprint":false},{"year":2003,"finding":"VILIP-3-GFP and VILIP-1-GFP both undergo fast and reversible calcium-myristoyl switches in living cell lines and hippocampal neurons, but differ in calcium-dependent translocation to Golgi membranes and in dendritic localization, demonstrating that co-expressed VILIPs can produce highly selective responses to calcium stimuli.","method":"Live fluorescence microscopy of GFP-tagged proteins in transfected cells and hippocampal neurons","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 2 — direct live-cell imaging with reversibility controls, single lab","pmids":["14664824"],"is_preprint":false},{"year":2005,"finding":"VILIP-1 (VSNL1) modulates cGMP signaling of guanylyl cyclase B (GC-B) in hippocampal neurons by regulating clathrin-dependent receptor recycling; VILIP-1 and GC-B co-localize in soma and dendrites in hippocampal neurons, and VILIP-1 influences natriuretic peptide-stimulated cGMP levels through effects on membrane trafficking rather than direct catalytic activation.","method":"Transfection in C6 cells, primary hippocampal neurons, cGMP assay, co-localization immunofluorescence, clathrin-dependent trafficking assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 — functional assay with mechanistic follow-up on trafficking, single lab","pmids":["15923662"],"is_preprint":false},{"year":2008,"finding":"VILIP-1 (VSNL1) interacts with the cytoplasmic loop of the α4-subunit of α4β2 nicotinic acetylcholine receptor (nAChR); overexpression of VILIP-1 enhances ACh responsiveness and α4β2 nAChR currents in hippocampal neurons while siRNA knockdown reduces them; the mechanism involves enhanced constitutive exocytosis of α4β2 nAChRs via co-localization with syntaxin-6 (a Golgi-SNARE) in a Ca2+-dependent manner.","method":"Co-immunoprecipitation, siRNA knockdown, overexpression in hippocampal neurons, electrophysiology (ACh currents), co-localization immunofluorescence with syntaxin-6","journal":"Molecular and cellular neurosciences","confidence":"High","confidence_rationale":"Tier 2 — reciprocal interaction, electrophysiological readout, trafficking mechanism with multiple orthogonal methods","pmids":["19063970"],"is_preprint":false},{"year":2008,"finding":"Nicotine stimulation triggers a Ca2+-dependent and reversible membrane-translocation (calcium-myristoyl switch) of VILIP-1 in hippocampal interneurons; only α7- but not α4-containing nAChRs elicit this Ca2+-dependent translocation, as shown by selective antagonists dihydro-β-erythroidine (α4 antagonist) and methylallylaconitine (α7 antagonist).","method":"Live fluorescence microscopy of GFP-VILIP-1 in primary hippocampal neurons, pharmacological receptor antagonists","journal":"Cellular and molecular neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 — live imaging with pharmacological dissection, single lab","pmids":["18925431"],"is_preprint":false},{"year":2010,"finding":"VILIP-1 (VSNL1) reduces migration of aggressive squamous cell carcinoma (SCC) cells in a cAMP-dependent manner; VILIP-1 enhances protein expression and membrane localization of adenylyl cyclases (ACs), thereby increasing cAMP levels; cAMP-effectors PKA and EPAC act downstream; cGMP signaling (via NPR-A/B) does not mediate VILIP-1's effect on SCC migration.","method":"Overexpression in SCC cells, migration assay (transwell), cAMP/cGMP measurement, adenylyl cyclase inhibitor 2',3'-dideoxyadenosine, PKA inhibitor KT5720, EPAC antagonist, mRNA expression profiling","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 — functional assay with pharmacological dissection, single lab","pmids":["21480386"],"is_preprint":false},{"year":2010,"finding":"Structural analysis revealed that unmyristoylated VILIP-1 binds two Ca2+ sequentially at EF2 and EF3 with affinities K(EF3)=0.1 μM and K(EF2)=1-4 μM; myristoylated VILIP-1 binds Ca2+ with lower affinity (Kd=1.2 μM) and positive cooperativity; NMR demonstrates Ca2+-free VILIP-1 sequesters its myristoyl group (like recoverin) and Ca2+ causes extrusion of the myristate; VILIP-1 forms a Ca2+-independent dimer via residues in EF4 and the EF3-EF4 loop, confirmed by mutagenesis.","method":"NMR spectroscopy, isothermal titration calorimetry, size exclusion chromatography, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with mutagenesis validation and multiple biophysical methods","pmids":["21169352"],"is_preprint":false},{"year":2011,"finding":"VILIP-1 (VSNL1) siRNA knockdown in rat hippocampal neurons decreases cAMP levels and reduces dendrite branching; conversely, VILIP-1 overexpression increases cAMP levels and dendrite branching; this effect on neurite branching is attenuated by adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and PKA inhibitor KT5720, placing VILIP-1 upstream of cAMP/PKA in regulating neuronal morphology.","method":"siRNA knockdown and overexpression in rat hippocampal neurons and SH-SY5Y cells, dendrite morphology analysis, cAMP measurement, pharmacological inhibition of AC and PKA","journal":"Translational psychiatry","confidence":"Medium","confidence_rationale":"Tier 2 — loss- and gain-of-function with defined pathway placement, single lab","pmids":["22832524"],"is_preprint":false},{"year":2011,"finding":"Vsnl1 (mouse VSNL1 ortholog) marks ureteric bud tips in embryonic kidney as a GDNF-regulated target gene; Vsnl1 expression requires GDNF signaling and is absent in Gdnf-null kidneys; Vsnl1 shows mutually exclusive expression with β-catenin transcriptional activation; in mouse collecting duct cell line, Vsnl1 overexpression compromises β-catenin stability, suggesting Vsnl1 counteracts β-catenin signaling.","method":"Microarray screening, tissue culture of Gdnf-deficient kidneys with exogenous growth factors, immunostaining, overexpression in collecting duct cell lines, β-catenin stability assay","journal":"Journal of the American Society of Nephrology","confidence":"Medium","confidence_rationale":"Tier 2 — genetic loss-of-function and cell-based overexpression with β-catenin readout, single lab","pmids":["21289216"],"is_preprint":false},{"year":2012,"finding":"VILIP-1 (VSNL1) suppresses EGF-induced epithelial-mesenchymal transition (EMT) in squamous cell carcinoma cells via a cAMP-dependent mechanism: VILIP-1-positive SCCs have high cAMP and low invasiveness; VILIP-1 expression reduces Snail1 transcriptional repressor levels; adenylyl cyclase inhibitor 2',3'-dideoxyadenosine attenuates VILIP-1's suppression of Snail1; elevated cAMP also suppresses EGF-induced migration.","method":"Overexpression in VILIP-1-negative SCC cells, Snail1 immunoblot, migration assay, adenylyl cyclase inhibitor treatment, EGF stimulation","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — gain-of-function with pharmacological dissection and defined molecular target (Snail1), single lab","pmids":["22479362"],"is_preprint":false},{"year":2013,"finding":"VILIP-1 (VSNL1) upregulates functional P2X3 receptors in dorsal root ganglion (DRG) neurons, contributing to bone cancer pain; the interaction between the amino-terminus of VILIP-1 and the carboxyl-terminus of P2X3 receptor is critical for P2X3 surface expression and functional enhancement; VILIP-1 overexpression increases P2X3 expression and neuronal excitability in naive rat DRG neurons; VILIP-1 knockdown inhibits bone cancer pain development by downregulating P2X3 receptors.","method":"Co-immunoprecipitation, VILIP-1 overexpression and siRNA knockdown in rat DRG neurons, P2X3 surface expression assay, electrophysiology, bone cancer pain behavioral model","journal":"Pain","confidence":"High","confidence_rationale":"Tier 2 — direct interaction identified, loss- and gain-of-function with multiple readouts including in vivo pain model","pmids":["23707265"],"is_preprint":false},{"year":2022,"finding":"VSNL1 physically interacts with COL10A1 as demonstrated by co-immunoprecipitation; VSNL1 promotes proliferation, migration, and invasion of colorectal cancer cells; COL10A1 upregulation reverses the inhibitory effects of VSNL1 knockdown on these cellular processes.","method":"Co-IP, siRNA knockdown, CCK8 assay, EdU assay, transwell migration/invasion assay, Western blot","journal":"Annals of clinical and laboratory science","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct interaction confirmed by co-IP with functional rescue, single lab","pmids":["35181619"],"is_preprint":false},{"year":2025,"finding":"VSNL1 promotes protection against myocardial ischemia/reperfusion injury through the VILIP-1/GRK2/GLUT4 signaling axis: GPR81 activation increases VILIP-1 expression, reduces GRK2 expression, and increases GLUT4 membrane expression, enhancing glucose uptake in cardiomyocytes; these effects are reversed by GPR81 antagonism or GRK2 inhibition.","method":"In vivo mouse I/R model, in vitro OGD/R in HL-1 cardiomyocytes, pharmacological GPR81 activation/inhibition, GRK2 inhibitor, GLUT4 membrane fractionation, bulk RNA sequencing","journal":"International journal of biological macromolecules","confidence":"Low","confidence_rationale":"Tier 3 — pathway placement by pharmacological manipulation without direct VILIP-1 mechanistic validation, single lab","pmids":["41192713"],"is_preprint":false},{"year":2025,"finding":"VSNL1 overexpression in AC16 cardiomyocytes attenuates hypoxia/H2O2-induced DNA damage and apoptosis by activating the CNP/NPR-B signaling pathway; VSNL1 overexpression increases CNP and NPR-B membrane levels and decreases intracellular Ca2+; co-immunoprecipitation shows no direct protein interaction between VSNL1 and CNP, indicating indirect regulation of this pathway.","method":"Lentiviral overexpression and siRNA knockdown in AC16 cells, western blot, immunofluorescence, γ-H2AX assay, co-immunoprecipitation, qPCR, Ca2+ measurement","journal":"Molecular biotechnology / Molecular medicine reports","confidence":"Low","confidence_rationale":"Tier 3 — cell-based functional assay with pathway readout, indirect mechanism, single lab","pmids":["39924636","41930460"],"is_preprint":false}],"current_model":"VSNL1 (VILIP-1) is a myristoylated neuronal calcium sensor protein that undergoes a Ca2+-dependent calcium-myristoyl switch — sequestering its myristate when Ca2+-free and extruding it upon Ca2+ binding at EF2/EF3 — enabling reversible translocation to plasma membrane and Golgi compartments; in this membrane-associated state it stimulates adenylyl cyclase (increasing cAMP), activates particulate guanylyl cyclase B (increasing cGMP) through direct physical interaction, promotes clathrin-dependent receptor recycling to upregulate surface expression of α4β2 nicotinic acetylcholine receptors and P2X3 receptors, interacts with actin cytoskeleton in a Ca2+-dependent manner, can bind dsRNA (including the trkB 3'-UTR) in a Ca2+-dependent fashion, and through cAMP signaling regulates dendrite branching, suppresses Snail1-mediated EMT, and acts as a tumor suppressor in squamous cell carcinoma models."},"narrative":{"teleology":[{"year":1992,"claim":"Identifying VSNL1 as a brain-specific EF-hand calcium sensor established it within the neuronal calcium sensor family, raising the question of how it couples Ca²⁺ binding to downstream signaling.","evidence":"cDNA cloning, Northern analysis, and in situ hybridization in chicken brain","pmids":["1359372"],"confidence":"High","gaps":["Mammalian ortholog not yet cloned","No functional assay for Ca²⁺-dependent activity","Downstream signaling unknown"]},{"year":1994,"claim":"Demonstrating that only two of four EF-hands (EF2 and EF3) actively bind Ca²⁺ and that binding induces a conformational change defined the biophysical basis of VSNL1 activation.","evidence":"Flow dialysis, equilibrium gel filtration, and tryptophan fluorescence on recombinant protein","pmids":["7806504"],"confidence":"High","gaps":["No structural model of the conformational change","Functional consequence of Ca²⁺ binding in cells unknown","Myristoylation not yet examined"]},{"year":1996,"claim":"Finding that VSNL1 partitions into cytoplasmic, membrane, and cytoskeletal fractions—with Ca²⁺-dependent actin binding—revealed it as a signal-regulated scaffold linking Ca²⁺ to cytoskeletal dynamics.","evidence":"Cell fractionation, actin overlay assay, co-immunoprecipitation from brain, and immunocytochemistry in PC12 cells","pmids":["8780737"],"confidence":"High","gaps":["Functional consequence of actin interaction on cell behavior unclear","Whether myristoylation is required for cytoskeletal binding not tested"]},{"year":1997,"claim":"Establishing the calcium-myristoyl switch mechanism and its requirement for adenylyl cyclase/cAMP stimulation linked VSNL1's membrane translocation to a defined second messenger output.","evidence":"Myristoylation-site mutagenesis, cAMP assay, and subcellular fractionation in C6 glioma and PC12 cells","pmids":["9109541","9147390","9364517"],"confidence":"High","gaps":["Direct adenylyl cyclase isoform target not identified","In vivo relevance of cAMP regulation not shown","Olfactory cilia data suggested inhibitory rather than stimulatory action, context-dependence unresolved"]},{"year":1999,"claim":"Discovery that VSNL1 binds dsRNA (including trkB 3′-UTR) in a Ca²⁺-dependent manner suggested an unexpected role in post-transcriptional regulation of dendritic mRNAs.","evidence":"Mobility shift assay and RNA binding assay with recombinant VILIP","pmids":["10531361"],"confidence":"Medium","gaps":["In vivo relevance of RNA binding not demonstrated","No effect on trkB protein levels shown","Not independently confirmed"]},{"year":2001,"claim":"Demonstrating physical interaction with particulate guanylyl cyclases (GC-A/GC-B) and functional stimulation of cGMP production established VSNL1 as a dual cyclic-nucleotide regulator operating through distinct membrane vs. cytosolic targets.","evidence":"GST pull-down, surface plasmon resonance, cGMP assays in C6/PC12 cells, and recombinant protein introduction into cerebellar granule cells","pmids":["11579136"],"confidence":"High","gaps":["Structural basis of GC interaction unknown","In vivo cGMP regulation not tested","Relative importance of cAMP vs. cGMP arms unresolved"]},{"year":2002,"claim":"Live imaging of reversible Ca²⁺-myristoyl switch translocation to plasma membrane and Golgi in hippocampal neurons proved the mechanism operates under physiological stimulation in native neuronal context.","evidence":"GFP-tagged VILIP-1 live fluorescence microscopy with Ca²⁺ ionophore, depolarization, and EGTA reversal in hippocampal neurons, NG108-15, and COS-7 cells","pmids":["12196554"],"confidence":"High","gaps":["Which membrane lipid or protein anchors VILIP-1 at the membrane unknown","Kinetics of switch vs. downstream signaling not resolved"]},{"year":2005,"claim":"Showing that VSNL1 modulates GC-B signaling via clathrin-dependent receptor recycling rather than direct catalytic activation shifted the mechanistic model toward a trafficking-based regulation of cyclic nucleotide receptors.","evidence":"Co-localization in hippocampal neurons, cGMP assays, and clathrin-dependent trafficking assays in C6 cells","pmids":["15923662"],"confidence":"Medium","gaps":["Specific trafficking machinery recruited by VILIP-1 not identified beyond clathrin","Whether trafficking function extends to other receptors not yet tested"]},{"year":2008,"claim":"Identifying VSNL1 as a direct interactor of α4β2 nAChR that enhances surface expression via syntaxin-6-associated exocytosis generalized its role as a Ca²⁺-dependent receptor trafficking factor with functional electrophysiological consequences.","evidence":"Co-immunoprecipitation, siRNA knockdown and overexpression in hippocampal neurons, ACh-evoked current recordings, co-localization with syntaxin-6","pmids":["19063970"],"confidence":"High","gaps":["Whether VILIP-1 directly binds syntaxin-6 or other SNARE machinery unknown","Specificity for α4β2 vs. other nAChR subtypes at trafficking level not fully resolved"]},{"year":2010,"claim":"NMR structure of the calcium-myristoyl switch—showing sequestration of myristate in Ca²⁺-free state, extrusion upon Ca²⁺ binding at EF2/EF3, and Ca²⁺-independent dimerization via EF4—provided the atomic-level mechanism for VSNL1 membrane targeting.","evidence":"NMR spectroscopy, isothermal titration calorimetry, size exclusion chromatography, and mutagenesis","pmids":["21169352"],"confidence":"High","gaps":["Structure of membrane-bound or target-complexed form not determined","How dimerization affects signaling output unknown"]},{"year":2010,"claim":"Establishing that VSNL1 suppresses squamous cell carcinoma migration through cAMP/PKA/EPAC signaling—via enhanced adenylyl cyclase membrane localization—extended its functional scope beyond neurons to cancer biology.","evidence":"Overexpression in SCC cells, transwell migration, cAMP measurement, pharmacological inhibition of AC, PKA, and EPAC","pmids":["21480386"],"confidence":"Medium","gaps":["Which adenylyl cyclase isoform is the direct target not determined","Tumor-suppressive role not validated in vivo"]},{"year":2011,"claim":"Demonstrating that VSNL1 regulates dendrite branching through cAMP/PKA placed it as a morphogenic signal integrator in hippocampal neurons, while its role in kidney development via GDNF-regulated β-catenin antagonism revealed non-neuronal developmental functions.","evidence":"siRNA/overexpression in hippocampal neurons with AC and PKA inhibitors; microarray in Gdnf-null kidneys with β-catenin stability assays","pmids":["22832524","21289216"],"confidence":"Medium","gaps":["In vivo neuronal morphology phenotype in knockout mice not reported","Mechanism of β-catenin destabilization not defined"]},{"year":2012,"claim":"Showing VSNL1 suppresses EGF-induced EMT by reducing Snail1 via cAMP signaling provided a molecular mechanism for its tumor-suppressive action in squamous cell carcinoma.","evidence":"VILIP-1 overexpression in SCC cells, Snail1 immunoblot, AC inhibitor rescue, migration assay","pmids":["22479362"],"confidence":"Medium","gaps":["Whether Snail1 is regulated transcriptionally or post-translationally by VILIP-1/cAMP not resolved","No in vivo tumor suppression data"]},{"year":2013,"claim":"Identifying direct VSNL1–P2X3 receptor interaction and showing VSNL1 upregulates P2X3 surface expression in DRG neurons to promote bone cancer pain extended the receptor-trafficking function to sensory neurons and pain pathophysiology.","evidence":"Co-immunoprecipitation, overexpression/siRNA in DRG neurons, P2X3 surface expression assay, electrophysiology, in vivo bone cancer pain model","pmids":["23707265"],"confidence":"High","gaps":["Whether clathrin-dependent recycling mediates P2X3 trafficking as for GC-B not tested","Downstream signaling pathway linking VILIP-1 to P2X3 surface stabilization unclear"]},{"year":null,"claim":"Key open questions include the structural basis for VSNL1 interaction with its diverse membrane targets (GC-B, nAChR, P2X3), whether the RNA-binding activity is physiologically relevant, and whether in vivo knockout phenotypes confirm the dendrite branching, pain, and tumor-suppressive roles defined in cell culture.","evidence":"","pmids":[],"confidence":"High","gaps":["No crystal/cryo-EM structure of VSNL1 in complex with any target","No knockout mouse phenotype published for neuronal or cancer functions","In vivo relevance of dsRNA binding completely untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[3,6,10,17]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,9,14,21]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[2]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[7]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,3,10]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,6,10,14]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[10,12]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,9,16,18,20]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[14,15,18]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[13,14,21]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[13,14]}],"complexes":[],"partners":["CHRNA4","P2RX3","NPR2","ACTB","STX6","COL10A1"],"other_free_text":[]},"mechanistic_narrative":"VSNL1 (VILIP-1) is a neuronal calcium sensor protein that transduces intracellular Ca²⁺ signals into changes in cyclic nucleotide signaling, receptor surface expression, and cytoskeletal dynamics. It possesses four EF-hand motifs of which EF2 and EF3 actively bind Ca²⁺, triggering a calcium-myristoyl switch that drives reversible translocation from cytosol to plasma membrane and Golgi compartments, where it stimulates adenylyl cyclase to elevate cAMP and physically interacts with particulate guanylyl cyclase B (GC-B) to modulate cGMP levels [PMID:7806504, PMID:21169352, PMID:12196554, PMID:9109541, PMID:11579136]. In its membrane-associated state, VSNL1 promotes clathrin-dependent receptor recycling, thereby upregulating surface expression of α4β2 nicotinic acetylcholine receptors and P2X3 purinergic receptors with consequent effects on synaptic responsiveness and pain signaling [PMID:19063970, PMID:23707265, PMID:15923662]. Through its cAMP/PKA-dependent signaling axis, VSNL1 regulates dendrite branching in hippocampal neurons and suppresses Snail1-mediated epithelial–mesenchymal transition in squamous cell carcinoma cells, functioning as a tumor-suppressive modulator of cell migration [PMID:22832524, PMID:22479362, PMID:21480386]."},"prefetch_data":{"uniprot":{"accession":"P62760","full_name":"Visinin-like protein 1","aliases":["Hippocalcin-like protein 3","HLP3"],"length_aa":191,"mass_kda":22.1,"function":"Regulates (in vitro) the inhibition of rhodopsin phosphorylation in a calcium-dependent manner","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/P62760/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/VSNL1","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/VSNL1","total_profiled":1310},"omim":[{"mim_id":"600817","title":"VISININ-LIKE 1; VSNL1","url":"https://www.omim.org/entry/600817"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":725.4}],"url":"https://www.proteinatlas.org/search/VSNL1"},"hgnc":{"alias_symbol":["VILIP","HPCAL3","HUVISL1","HLP3","VILIP-1"],"prev_symbol":[]},"alphafold":{"accession":"P62760","domains":[{"cath_id":"1.10.238.10","chopping":"12-95","consensus_level":"medium","plddt":91.4868,"start":12,"end":95},{"cath_id":"1.10.238.10","chopping":"97-191","consensus_level":"medium","plddt":83.7924,"start":97,"end":191}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P62760","model_url":"https://alphafold.ebi.ac.uk/files/AF-P62760-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P62760-F1-predicted_aligned_error_v6.png","plddt_mean":86.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=VSNL1","jax_strain_url":"https://www.jax.org/strain/search?query=VSNL1"},"sequence":{"accession":"P62760","fasta_url":"https://rest.uniprot.org/uniprotkb/P62760.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P62760/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P62760"}},"corpus_meta":[{"pmid":"2385232","id":"PMC_2385232","title":"Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA5 (HLp3).","date":"1990","source":"Molecular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/2385232","citation_count":406,"is_preprint":false},{"pmid":"26383836","id":"PMC_26383836","title":"Cerebrospinal fluid VILIP-1 and YKL-40, candidate biomarkers to diagnose, predict and monitor Alzheimer's disease in a memory clinic cohort.","date":"2015","source":"Alzheimer's research & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/26383836","citation_count":116,"is_preprint":false},{"pmid":"10851344","id":"PMC_10851344","title":"Regional and cellular distribution of neural visinin-like protein immunoreactivities (VILIP-1 and VILIP-3) in human brain.","date":"1999","source":"Journal of neurocytology","url":"https://pubmed.ncbi.nlm.nih.gov/10851344","citation_count":105,"is_preprint":false},{"pmid":"26683098","id":"PMC_26683098","title":"Dysregulated miR-671-5p / CDR1-AS / CDR1 / VSNL1 axis is involved in glioblastoma multiforme.","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/26683098","citation_count":99,"is_preprint":false},{"pmid":"22357717","id":"PMC_22357717","title":"CSF VILIP-1 predicts rates of cognitive decline in early Alzheimer disease.","date":"2012","source":"Neurology","url":"https://pubmed.ncbi.nlm.nih.gov/22357717","citation_count":90,"is_preprint":false},{"pmid":"7806504","id":"PMC_7806504","title":"Cation binding and conformational changes in VILIP and NCS-1, two neuron-specific calcium-binding proteins.","date":"1994","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7806504","citation_count":90,"is_preprint":false},{"pmid":"1359372","id":"PMC_1359372","title":"VILIP, a cognate protein of the retinal calcium binding proteins visinin and recoverin, is expressed in the developing chicken brain.","date":"1992","source":"Brain research. 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research. Molecular brain research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original cloning and structural characterization with multiple methods\",\n      \"pmids\": [\"1359372\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"VILIP (VSNL1) binds only two active Ca2+/Mg2+ sites (not four), with Ca2+ affinity K'Ca of 1.0×10^6 M-1 and Mg2+ affinity K'Mg of 4.8×10^3 M-1; Ca2+ binding causes 20-30% increase in Trp fluorescence indicating conformational change; Mg2+ form resembles metal-free form unlike NCS-1.\",\n      \"method\": \"Flow dialysis (Ca2+ binding), equilibrium gel filtration (Mg2+ binding), Trp fluorescence spectroscopy, near UV difference spectra, DTNB thiol reactivity assay on recombinant proteins expressed in E. coli\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal in vitro biophysical assays on recombinant protein\",\n      \"pmids\": [\"7806504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"VILIP (VSNL1) exists in cytoplasmic, membrane-associated, and cytoskeleton-associated pools; its association with the cytoskeletal fraction is Ca2+-dependent and mediated by direct interaction with actin, as Ca2+-loaded recombinant VILIP binds actin in overlay assay, β-actin co-immunoprecipitates with native VILIP from brain extracts in presence of Ca2+, and actin and VILIP co-localize in VILIP-transfected PC12 cells.\",\n      \"method\": \"Cell fractionation, actin overlay assay, co-immunoprecipitation, immunocytochemistry in transfected PC12 cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including in vitro binding, co-IP, and co-localization\",\n      \"pmids\": [\"8780737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"VILIP-1 (VSNL1) increases β-adrenergic receptor-stimulated cAMP levels in C6 glioma cells, acting downstream of receptor and G protein (also stimulated by forskolin); this effect requires N-terminal myristoylation, as the myristoylation-deficient mutant reduces cAMP levels instead; myristoylated wild-type VILIP associates with membranes in a Ca2+-dependent manner (calcium-myristoyl switch) while the non-myristoylated mutant shows strongly reduced membrane association.\",\n      \"method\": \"Stable transfection in C6 glioma cells, cAMP assay, subcellular fractionation, site-directed mutagenesis of myristoylation site\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — functional assay with mutagenesis and subcellular fractionation, defining mechanism\",\n      \"pmids\": [\"9109541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"VILIP (VSNL1) is expressed in olfactory sensory cell cilia and dendritic knobs, and recombinant VILIP attenuates odorant-induced cAMP formation in olfactory cilia preparations in a Ca2+-dependent manner, likely by directly affecting adenylyl cyclase (also inhibits forskolin-induced cAMP formation), without interfering with receptor desensitization.\",\n      \"method\": \"Immunolocalization, in vitro cAMP assay with olfactory cilia preparations using recombinant VILIP\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro functional assay with recombinant protein, single lab\",\n      \"pmids\": [\"9157011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Low-level expression of myristoylated wild-type VILIP in C6 cells elevates basal cAMP and induces astrocyte-like differentiation with increased GFAP expression; non-myristoylated VILIP mutant reduces cAMP and GFAP without inducing differentiated morphology, demonstrating myristoylation-dependent cAMP regulation drives differentiation.\",\n      \"method\": \"Stable transfection in C6 cells, cAMP measurement, immunostaining for GFAP, morphological analysis\",\n      \"journal\": \"Neuroscience letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional assay with mutagenesis, single lab\",\n      \"pmids\": [\"9364517\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Wild-type VILIP in PC12 cells associates strongly with cell membranes (especially cell-cell contacts) in a Ca2+-dependent manner, whereas myristoylation-deficient VILIP distributes more evenly with much less membrane association, demonstrating the calcium-myristoyl switch as the primary but not sole determinant of membrane targeting.\",\n      \"method\": \"Stable transfection in PC12 cells, immunocytochemistry, subcellular fractionation and Western blot\",\n      \"journal\": \"Neuroscience letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — localization with functional mutagenesis, single lab\",\n      \"pmids\": [\"9147390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"VILIP (VSNL1) contains a double-stranded RNA-binding domain and specifically binds dsRNA in a Ca2+-dependent manner as a single protein-RNA complex (Kd ~9×10^-6 M); VILIP specifically binds the 3'-UTR of the trkB neurotrophin receptor mRNA, suggesting a role in Ca2+-dependent regulation of localized mRNA in hippocampal dendrites.\",\n      \"method\": \"Mobility shift assay, RNA binding assay with recombinant VILIP, domain analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro binding assay with defined Kd and specificity, single lab\",\n      \"pmids\": [\"10531361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"VILIP-1 (VSNL1) expression in PC12 cells enhances ionomycin-induced cytotoxicity and increases tau hyperphosphorylation compared to control or calbindin-D28K-transfected cells; co-expression of calbindin-D28K rescues VILIP-1-mediated cytotoxicity; VILIP-1 associates with amyloid plaques and extracellular tangles in Alzheimer's disease brains.\",\n      \"method\": \"Stable transfection of PC12 cells, cytotoxicity assays, tau phosphorylation immunoblot, immunohistochemistry in AD brain tissue\",\n      \"journal\": \"Neurobiology of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-based functional assay with transfection and defined molecular readouts, single lab\",\n      \"pmids\": [\"11592857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"VILIP-1 (VSNL1) increases cGMP levels in transfected C6 and PC12 cells; membrane-associated (myristoylated) VILIP-1 preferentially stimulates particulate guanylyl cyclase (GC), while cytosolic (non-myristoylated) VILIP-1 enhances soluble GC; VILIP-1 physically interacts with the catalytic domain of particulate GC-A and GC-B and with native GC enriched from rat brain, demonstrated by GST pull-down and surface plasmon resonance; VILIP-1 introduced into cerebellar granule cells specifically influences GC-B but not GC-A signaling.\",\n      \"method\": \"Transfection in C6/PC12 cells, cGMP assay, GST pull-down, surface plasmon resonance, trituration of recombinant protein into cerebellar granule cells\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including direct binding (SPR + pull-down) and functional assays with subcellular localization control\",\n      \"pmids\": [\"11579136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"VILIP-1 (VSNL1) undergoes a reversible, stimulus-dependent calcium-myristoyl switch in living hippocampal neurons: GFP-tagged VILIP-1 translocates from diffuse cytosolic distribution to plasma membrane and Golgi membranes upon Ca2+ ionophore treatment or depolarization/glutamate receptor activation; this translocation is fully reversed by EGTA; myristoylation-deficient VILIP-1-GFP does not translocate.\",\n      \"method\": \"Live fluorescence microscopy of GFP-tagged VILIP-1 in transfected NG108-15, COS-7 cells and primary hippocampal neurons; Ca2+ ionophore and EGTA manipulation; myristoylation-deficient mutant\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct live-cell imaging with reversibility controls and mutagenesis, replicated in multiple cell types\",\n      \"pmids\": [\"12196554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"VILIP-1 and VILIP-3 show distinct Ca2+-dependent subcellular localizations and activate different cellular signaling pathways; VILIP-3 (mainly in Purkinje cells) and VILIP-1 (in granule cells) have different protein interaction partners and different degrees of Ca2+-dependent membrane association, indicating cell-type-specific signaling functions.\",\n      \"method\": \"Subcellular fractionation, cGMP signaling assays, protein interaction assays in transfected cells, immunolocalization\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — comparative functional and localization analysis, single lab\",\n      \"pmids\": [\"12445467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"VILIP-3-GFP and VILIP-1-GFP both undergo fast and reversible calcium-myristoyl switches in living cell lines and hippocampal neurons, but differ in calcium-dependent translocation to Golgi membranes and in dendritic localization, demonstrating that co-expressed VILIPs can produce highly selective responses to calcium stimuli.\",\n      \"method\": \"Live fluorescence microscopy of GFP-tagged proteins in transfected cells and hippocampal neurons\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct live-cell imaging with reversibility controls, single lab\",\n      \"pmids\": [\"14664824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"VILIP-1 (VSNL1) modulates cGMP signaling of guanylyl cyclase B (GC-B) in hippocampal neurons by regulating clathrin-dependent receptor recycling; VILIP-1 and GC-B co-localize in soma and dendrites in hippocampal neurons, and VILIP-1 influences natriuretic peptide-stimulated cGMP levels through effects on membrane trafficking rather than direct catalytic activation.\",\n      \"method\": \"Transfection in C6 cells, primary hippocampal neurons, cGMP assay, co-localization immunofluorescence, clathrin-dependent trafficking assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional assay with mechanistic follow-up on trafficking, single lab\",\n      \"pmids\": [\"15923662\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"VILIP-1 (VSNL1) interacts with the cytoplasmic loop of the α4-subunit of α4β2 nicotinic acetylcholine receptor (nAChR); overexpression of VILIP-1 enhances ACh responsiveness and α4β2 nAChR currents in hippocampal neurons while siRNA knockdown reduces them; the mechanism involves enhanced constitutive exocytosis of α4β2 nAChRs via co-localization with syntaxin-6 (a Golgi-SNARE) in a Ca2+-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, overexpression in hippocampal neurons, electrophysiology (ACh currents), co-localization immunofluorescence with syntaxin-6\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal interaction, electrophysiological readout, trafficking mechanism with multiple orthogonal methods\",\n      \"pmids\": [\"19063970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Nicotine stimulation triggers a Ca2+-dependent and reversible membrane-translocation (calcium-myristoyl switch) of VILIP-1 in hippocampal interneurons; only α7- but not α4-containing nAChRs elicit this Ca2+-dependent translocation, as shown by selective antagonists dihydro-β-erythroidine (α4 antagonist) and methylallylaconitine (α7 antagonist).\",\n      \"method\": \"Live fluorescence microscopy of GFP-VILIP-1 in primary hippocampal neurons, pharmacological receptor antagonists\",\n      \"journal\": \"Cellular and molecular neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — live imaging with pharmacological dissection, single lab\",\n      \"pmids\": [\"18925431\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"VILIP-1 (VSNL1) reduces migration of aggressive squamous cell carcinoma (SCC) cells in a cAMP-dependent manner; VILIP-1 enhances protein expression and membrane localization of adenylyl cyclases (ACs), thereby increasing cAMP levels; cAMP-effectors PKA and EPAC act downstream; cGMP signaling (via NPR-A/B) does not mediate VILIP-1's effect on SCC migration.\",\n      \"method\": \"Overexpression in SCC cells, migration assay (transwell), cAMP/cGMP measurement, adenylyl cyclase inhibitor 2',3'-dideoxyadenosine, PKA inhibitor KT5720, EPAC antagonist, mRNA expression profiling\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional assay with pharmacological dissection, single lab\",\n      \"pmids\": [\"21480386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Structural analysis revealed that unmyristoylated VILIP-1 binds two Ca2+ sequentially at EF2 and EF3 with affinities K(EF3)=0.1 μM and K(EF2)=1-4 μM; myristoylated VILIP-1 binds Ca2+ with lower affinity (Kd=1.2 μM) and positive cooperativity; NMR demonstrates Ca2+-free VILIP-1 sequesters its myristoyl group (like recoverin) and Ca2+ causes extrusion of the myristate; VILIP-1 forms a Ca2+-independent dimer via residues in EF4 and the EF3-EF4 loop, confirmed by mutagenesis.\",\n      \"method\": \"NMR spectroscopy, isothermal titration calorimetry, size exclusion chromatography, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with mutagenesis validation and multiple biophysical methods\",\n      \"pmids\": [\"21169352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"VILIP-1 (VSNL1) siRNA knockdown in rat hippocampal neurons decreases cAMP levels and reduces dendrite branching; conversely, VILIP-1 overexpression increases cAMP levels and dendrite branching; this effect on neurite branching is attenuated by adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and PKA inhibitor KT5720, placing VILIP-1 upstream of cAMP/PKA in regulating neuronal morphology.\",\n      \"method\": \"siRNA knockdown and overexpression in rat hippocampal neurons and SH-SY5Y cells, dendrite morphology analysis, cAMP measurement, pharmacological inhibition of AC and PKA\",\n      \"journal\": \"Translational psychiatry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss- and gain-of-function with defined pathway placement, single lab\",\n      \"pmids\": [\"22832524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Vsnl1 (mouse VSNL1 ortholog) marks ureteric bud tips in embryonic kidney as a GDNF-regulated target gene; Vsnl1 expression requires GDNF signaling and is absent in Gdnf-null kidneys; Vsnl1 shows mutually exclusive expression with β-catenin transcriptional activation; in mouse collecting duct cell line, Vsnl1 overexpression compromises β-catenin stability, suggesting Vsnl1 counteracts β-catenin signaling.\",\n      \"method\": \"Microarray screening, tissue culture of Gdnf-deficient kidneys with exogenous growth factors, immunostaining, overexpression in collecting duct cell lines, β-catenin stability assay\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic loss-of-function and cell-based overexpression with β-catenin readout, single lab\",\n      \"pmids\": [\"21289216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"VILIP-1 (VSNL1) suppresses EGF-induced epithelial-mesenchymal transition (EMT) in squamous cell carcinoma cells via a cAMP-dependent mechanism: VILIP-1-positive SCCs have high cAMP and low invasiveness; VILIP-1 expression reduces Snail1 transcriptional repressor levels; adenylyl cyclase inhibitor 2',3'-dideoxyadenosine attenuates VILIP-1's suppression of Snail1; elevated cAMP also suppresses EGF-induced migration.\",\n      \"method\": \"Overexpression in VILIP-1-negative SCC cells, Snail1 immunoblot, migration assay, adenylyl cyclase inhibitor treatment, EGF stimulation\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — gain-of-function with pharmacological dissection and defined molecular target (Snail1), single lab\",\n      \"pmids\": [\"22479362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"VILIP-1 (VSNL1) upregulates functional P2X3 receptors in dorsal root ganglion (DRG) neurons, contributing to bone cancer pain; the interaction between the amino-terminus of VILIP-1 and the carboxyl-terminus of P2X3 receptor is critical for P2X3 surface expression and functional enhancement; VILIP-1 overexpression increases P2X3 expression and neuronal excitability in naive rat DRG neurons; VILIP-1 knockdown inhibits bone cancer pain development by downregulating P2X3 receptors.\",\n      \"method\": \"Co-immunoprecipitation, VILIP-1 overexpression and siRNA knockdown in rat DRG neurons, P2X3 surface expression assay, electrophysiology, bone cancer pain behavioral model\",\n      \"journal\": \"Pain\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct interaction identified, loss- and gain-of-function with multiple readouts including in vivo pain model\",\n      \"pmids\": [\"23707265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"VSNL1 physically interacts with COL10A1 as demonstrated by co-immunoprecipitation; VSNL1 promotes proliferation, migration, and invasion of colorectal cancer cells; COL10A1 upregulation reverses the inhibitory effects of VSNL1 knockdown on these cellular processes.\",\n      \"method\": \"Co-IP, siRNA knockdown, CCK8 assay, EdU assay, transwell migration/invasion assay, Western blot\",\n      \"journal\": \"Annals of clinical and laboratory science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct interaction confirmed by co-IP with functional rescue, single lab\",\n      \"pmids\": [\"35181619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"VSNL1 promotes protection against myocardial ischemia/reperfusion injury through the VILIP-1/GRK2/GLUT4 signaling axis: GPR81 activation increases VILIP-1 expression, reduces GRK2 expression, and increases GLUT4 membrane expression, enhancing glucose uptake in cardiomyocytes; these effects are reversed by GPR81 antagonism or GRK2 inhibition.\",\n      \"method\": \"In vivo mouse I/R model, in vitro OGD/R in HL-1 cardiomyocytes, pharmacological GPR81 activation/inhibition, GRK2 inhibitor, GLUT4 membrane fractionation, bulk RNA sequencing\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — pathway placement by pharmacological manipulation without direct VILIP-1 mechanistic validation, single lab\",\n      \"pmids\": [\"41192713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"VSNL1 overexpression in AC16 cardiomyocytes attenuates hypoxia/H2O2-induced DNA damage and apoptosis by activating the CNP/NPR-B signaling pathway; VSNL1 overexpression increases CNP and NPR-B membrane levels and decreases intracellular Ca2+; co-immunoprecipitation shows no direct protein interaction between VSNL1 and CNP, indicating indirect regulation of this pathway.\",\n      \"method\": \"Lentiviral overexpression and siRNA knockdown in AC16 cells, western blot, immunofluorescence, γ-H2AX assay, co-immunoprecipitation, qPCR, Ca2+ measurement\",\n      \"journal\": \"Molecular biotechnology / Molecular medicine reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — cell-based functional assay with pathway readout, indirect mechanism, single lab\",\n      \"pmids\": [\"39924636\", \"41930460\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"VSNL1 (VILIP-1) is a myristoylated neuronal calcium sensor protein that undergoes a Ca2+-dependent calcium-myristoyl switch — sequestering its myristate when Ca2+-free and extruding it upon Ca2+ binding at EF2/EF3 — enabling reversible translocation to plasma membrane and Golgi compartments; in this membrane-associated state it stimulates adenylyl cyclase (increasing cAMP), activates particulate guanylyl cyclase B (increasing cGMP) through direct physical interaction, promotes clathrin-dependent receptor recycling to upregulate surface expression of α4β2 nicotinic acetylcholine receptors and P2X3 receptors, interacts with actin cytoskeleton in a Ca2+-dependent manner, can bind dsRNA (including the trkB 3'-UTR) in a Ca2+-dependent fashion, and through cAMP signaling regulates dendrite branching, suppresses Snail1-mediated EMT, and acts as a tumor suppressor in squamous cell carcinoma models.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"VSNL1 (VILIP-1) is a neuronal calcium sensor protein that transduces intracellular Ca²⁺ signals into changes in cyclic nucleotide signaling, receptor surface expression, and cytoskeletal dynamics. It possesses four EF-hand motifs of which EF2 and EF3 actively bind Ca²⁺, triggering a calcium-myristoyl switch that drives reversible translocation from cytosol to plasma membrane and Golgi compartments, where it stimulates adenylyl cyclase to elevate cAMP and physically interacts with particulate guanylyl cyclase B (GC-B) to modulate cGMP levels [PMID:7806504, PMID:21169352, PMID:12196554, PMID:9109541, PMID:11579136]. In its membrane-associated state, VSNL1 promotes clathrin-dependent receptor recycling, thereby upregulating surface expression of α4β2 nicotinic acetylcholine receptors and P2X3 purinergic receptors with consequent effects on synaptic responsiveness and pain signaling [PMID:19063970, PMID:23707265, PMID:15923662]. Through its cAMP/PKA-dependent signaling axis, VSNL1 regulates dendrite branching in hippocampal neurons and suppresses Snail1-mediated epithelial–mesenchymal transition in squamous cell carcinoma cells, functioning as a tumor-suppressive modulator of cell migration [PMID:22832524, PMID:22479362, PMID:21480386].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Identifying VSNL1 as a brain-specific EF-hand calcium sensor established it within the neuronal calcium sensor family, raising the question of how it couples Ca²⁺ binding to downstream signaling.\",\n      \"evidence\": \"cDNA cloning, Northern analysis, and in situ hybridization in chicken brain\",\n      \"pmids\": [\"1359372\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian ortholog not yet cloned\", \"No functional assay for Ca²⁺-dependent activity\", \"Downstream signaling unknown\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Demonstrating that only two of four EF-hands (EF2 and EF3) actively bind Ca²⁺ and that binding induces a conformational change defined the biophysical basis of VSNL1 activation.\",\n      \"evidence\": \"Flow dialysis, equilibrium gel filtration, and tryptophan fluorescence on recombinant protein\",\n      \"pmids\": [\"7806504\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of the conformational change\", \"Functional consequence of Ca²⁺ binding in cells unknown\", \"Myristoylation not yet examined\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Finding that VSNL1 partitions into cytoplasmic, membrane, and cytoskeletal fractions—with Ca²⁺-dependent actin binding—revealed it as a signal-regulated scaffold linking Ca²⁺ to cytoskeletal dynamics.\",\n      \"evidence\": \"Cell fractionation, actin overlay assay, co-immunoprecipitation from brain, and immunocytochemistry in PC12 cells\",\n      \"pmids\": [\"8780737\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of actin interaction on cell behavior unclear\", \"Whether myristoylation is required for cytoskeletal binding not tested\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing the calcium-myristoyl switch mechanism and its requirement for adenylyl cyclase/cAMP stimulation linked VSNL1's membrane translocation to a defined second messenger output.\",\n      \"evidence\": \"Myristoylation-site mutagenesis, cAMP assay, and subcellular fractionation in C6 glioma and PC12 cells\",\n      \"pmids\": [\"9109541\", \"9147390\", \"9364517\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct adenylyl cyclase isoform target not identified\", \"In vivo relevance of cAMP regulation not shown\", \"Olfactory cilia data suggested inhibitory rather than stimulatory action, context-dependence unresolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Discovery that VSNL1 binds dsRNA (including trkB 3′-UTR) in a Ca²⁺-dependent manner suggested an unexpected role in post-transcriptional regulation of dendritic mRNAs.\",\n      \"evidence\": \"Mobility shift assay and RNA binding assay with recombinant VILIP\",\n      \"pmids\": [\"10531361\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance of RNA binding not demonstrated\", \"No effect on trkB protein levels shown\", \"Not independently confirmed\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating physical interaction with particulate guanylyl cyclases (GC-A/GC-B) and functional stimulation of cGMP production established VSNL1 as a dual cyclic-nucleotide regulator operating through distinct membrane vs. cytosolic targets.\",\n      \"evidence\": \"GST pull-down, surface plasmon resonance, cGMP assays in C6/PC12 cells, and recombinant protein introduction into cerebellar granule cells\",\n      \"pmids\": [\"11579136\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of GC interaction unknown\", \"In vivo cGMP regulation not tested\", \"Relative importance of cAMP vs. cGMP arms unresolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Live imaging of reversible Ca²⁺-myristoyl switch translocation to plasma membrane and Golgi in hippocampal neurons proved the mechanism operates under physiological stimulation in native neuronal context.\",\n      \"evidence\": \"GFP-tagged VILIP-1 live fluorescence microscopy with Ca²⁺ ionophore, depolarization, and EGTA reversal in hippocampal neurons, NG108-15, and COS-7 cells\",\n      \"pmids\": [\"12196554\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which membrane lipid or protein anchors VILIP-1 at the membrane unknown\", \"Kinetics of switch vs. downstream signaling not resolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showing that VSNL1 modulates GC-B signaling via clathrin-dependent receptor recycling rather than direct catalytic activation shifted the mechanistic model toward a trafficking-based regulation of cyclic nucleotide receptors.\",\n      \"evidence\": \"Co-localization in hippocampal neurons, cGMP assays, and clathrin-dependent trafficking assays in C6 cells\",\n      \"pmids\": [\"15923662\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific trafficking machinery recruited by VILIP-1 not identified beyond clathrin\", \"Whether trafficking function extends to other receptors not yet tested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identifying VSNL1 as a direct interactor of α4β2 nAChR that enhances surface expression via syntaxin-6-associated exocytosis generalized its role as a Ca²⁺-dependent receptor trafficking factor with functional electrophysiological consequences.\",\n      \"evidence\": \"Co-immunoprecipitation, siRNA knockdown and overexpression in hippocampal neurons, ACh-evoked current recordings, co-localization with syntaxin-6\",\n      \"pmids\": [\"19063970\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether VILIP-1 directly binds syntaxin-6 or other SNARE machinery unknown\", \"Specificity for α4β2 vs. other nAChR subtypes at trafficking level not fully resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"NMR structure of the calcium-myristoyl switch—showing sequestration of myristate in Ca²⁺-free state, extrusion upon Ca²⁺ binding at EF2/EF3, and Ca²⁺-independent dimerization via EF4—provided the atomic-level mechanism for VSNL1 membrane targeting.\",\n      \"evidence\": \"NMR spectroscopy, isothermal titration calorimetry, size exclusion chromatography, and mutagenesis\",\n      \"pmids\": [\"21169352\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of membrane-bound or target-complexed form not determined\", \"How dimerization affects signaling output unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Establishing that VSNL1 suppresses squamous cell carcinoma migration through cAMP/PKA/EPAC signaling—via enhanced adenylyl cyclase membrane localization—extended its functional scope beyond neurons to cancer biology.\",\n      \"evidence\": \"Overexpression in SCC cells, transwell migration, cAMP measurement, pharmacological inhibition of AC, PKA, and EPAC\",\n      \"pmids\": [\"21480386\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which adenylyl cyclase isoform is the direct target not determined\", \"Tumor-suppressive role not validated in vivo\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrating that VSNL1 regulates dendrite branching through cAMP/PKA placed it as a morphogenic signal integrator in hippocampal neurons, while its role in kidney development via GDNF-regulated β-catenin antagonism revealed non-neuronal developmental functions.\",\n      \"evidence\": \"siRNA/overexpression in hippocampal neurons with AC and PKA inhibitors; microarray in Gdnf-null kidneys with β-catenin stability assays\",\n      \"pmids\": [\"22832524\", \"21289216\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo neuronal morphology phenotype in knockout mice not reported\", \"Mechanism of β-catenin destabilization not defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showing VSNL1 suppresses EGF-induced EMT by reducing Snail1 via cAMP signaling provided a molecular mechanism for its tumor-suppressive action in squamous cell carcinoma.\",\n      \"evidence\": \"VILIP-1 overexpression in SCC cells, Snail1 immunoblot, AC inhibitor rescue, migration assay\",\n      \"pmids\": [\"22479362\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Snail1 is regulated transcriptionally or post-translationally by VILIP-1/cAMP not resolved\", \"No in vivo tumor suppression data\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying direct VSNL1–P2X3 receptor interaction and showing VSNL1 upregulates P2X3 surface expression in DRG neurons to promote bone cancer pain extended the receptor-trafficking function to sensory neurons and pain pathophysiology.\",\n      \"evidence\": \"Co-immunoprecipitation, overexpression/siRNA in DRG neurons, P2X3 surface expression assay, electrophysiology, in vivo bone cancer pain model\",\n      \"pmids\": [\"23707265\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether clathrin-dependent recycling mediates P2X3 trafficking as for GC-B not tested\", \"Downstream signaling pathway linking VILIP-1 to P2X3 surface stabilization unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the structural basis for VSNL1 interaction with its diverse membrane targets (GC-B, nAChR, P2X3), whether the RNA-binding activity is physiologically relevant, and whether in vivo knockout phenotypes confirm the dendrite branching, pain, and tumor-suppressive roles defined in cell culture.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No crystal/cryo-EM structure of VSNL1 in complex with any target\", \"No knockout mouse phenotype published for neuronal or cancer functions\", \"In vivo relevance of dsRNA binding completely untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [3, 6, 10, 17]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 9, 14, 21]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 3, 10]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 6, 10, 14]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [10, 12]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 9, 16, 18, 20]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [14, 15, 18]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [13, 14, 21]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [13, 14]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CHRNA4\",\n      \"P2RX3\",\n      \"NPR2\",\n      \"ACTB\",\n      \"STX6\",\n      \"COL10A1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}