{"gene":"ACVR1B","run_date":"2026-06-09T22:02:40","timeline":{"discoveries":[{"year":1998,"finding":"ActRIB (ACVR1B) forms heteromeric complexes with type II activin receptors to mediate activin signaling; genetic knockout in mice shows it is required for egg cylinder organization and gastrulation, with chimera analysis demonstrating a role in primitive streak formation but not mesoderm differentiation per se.","method":"Gene targeting/knockout, chimera analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with defined developmental phenotype replicated across multiple chimera experiments in a single rigorous study","pmids":["9512518"],"is_preprint":false},{"year":1997,"finding":"Constitutively active ALK4 (ALK-4*) induces dose-dependent mesoderm including dorsal/anterior cell types (goosecoid, Xbra) and endoderm in Xenopus animal caps, whereas ALK-2* induces only ventral mesoderm; threshold responses to activin can be established by ALK4 signaling alone, and ventralizing ALK-2* signals antagonize ALK-4* dorsal signals.","method":"Constitutively active receptor mRNA injection, Xenopus animal cap assay, gene expression analysis","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — functional reconstitution with constitutively active and dominant-negative receptors, dose-response analysis, replicated across multiple experimental conditions","pmids":["9367435"],"is_preprint":false},{"year":1999,"finding":"The loop between kinase subdomains IV and V of ALK4 (seven amino acids) mediates its strong dorsal gene-inducing signaling specificity; transferring this loop from ALK4* to ALK2* confers dorsal gene induction on ALK2*, identifying this loop as a key determinant of ALK4 signaling specificity.","method":"Chimeric receptor construction, mRNA injection, Xenopus animal cap assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — chimeric mutagenesis with functional readout, single lab, single study","pmids":["10075688"],"is_preprint":false},{"year":2000,"finding":"Truncated ALK4 isoforms (Alk4-2, 4-3, 4-4) found in human pituitary tumors act as dominant-negative receptors: they co-immunoprecipitate with type II activin receptors but are not phosphorylated, blocking wild-type activin signaling and the antiproliferative effects of activin.","method":"Coimmunoprecipitation, reporter assays, stable transfection, cell proliferation assay","journal":"Molecular endocrinology (Baltimore, Md.)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus functional reporter and proliferation assays, single lab, multiple orthogonal methods","pmids":["11117535"],"is_preprint":false},{"year":2001,"finding":"ALK4 mediates Nodal signaling (Xnr1, mouse Nodal) only in the presence of the co-receptor Cripto; ALK4 can directly interact with Cripto. A dominant-negative ALK4 blocks all mesoderm-inducing ligands (Nodal, Xnr1, Xnr2, Xnr4, Activin), while dominant-negative ALK7 specifically blocks Nodal/Xnr1.","method":"Receptor reconstitution, Co-IP, dominant-negative receptor injection in Xenopus embryos","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — biochemical binding plus functional epistasis in multiple ligand/receptor combinations, replicated across Xenopus and mouse contexts","pmids":["11485994"],"is_preprint":false},{"year":2001,"finding":"ACVR1B harbors somatic mutations in pancreatic carcinoma, establishing it as a mutated tumor-suppressor gene in sporadic pancreatic cancer.","method":"Direct sequencing of pancreatic cancer samples","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct sequencing across multiple tumor samples, single lab, establishes somatic mutation status","pmids":["11248065"],"is_preprint":false},{"year":2002,"finding":"Cripto-1 binds directly to ALK4 on mammalian epithelial cells (identified by phage display and confirmed by co-immunoprecipitation and FACS); Cripto-1 phosphorylates Smad2 only in the presence of both Nodal and ALK4, while Cripto-1-stimulated MAPK and AKT activation is independent of Nodal and ALK4.","method":"Phage display library screening, coimmunoprecipitation, FACS, reporter assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal binding identified by two orthogonal methods (phage display + Co-IP), plus functional Smad2 phosphorylation assay, single lab","pmids":["11909953"],"is_preprint":false},{"year":2002,"finding":"SB-431542 inhibits ALK4, ALK5, and ALK7 kinase activity but has no effect on other ALK family members (ALK1, 2, 3, 6) or on ERK, JNK, or p38 MAP kinase pathways, making it a selective inhibitor of activin/TGF-β/Nodal type I receptor signaling via SMAD2/3.","method":"Biochemical kinase inhibition assay, reporter assays, cell signaling analysis","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assays plus multiple cell-based signaling readouts, widely replicated across many labs","pmids":["12065756"],"is_preprint":false},{"year":2003,"finding":"Five hydrophobic residues on the ALK4 extracellular domain (Leu40, Ile70, Val73, Leu75, Pro77) constitute a functional binding surface for activin-A; mutation of these residues to alanine substantially disrupts dominant-negative ALK4 activity and reduces 125I-activin-A crosslinking to ALK4 in the presence of ActRII.","method":"Site-directed mutagenesis, dominant-negative inhibition assay, 125I-activin crosslinking","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis combined with biochemical binding assay (crosslinking), multiple mutants tested, single lab","pmids":["12665502"],"is_preprint":false},{"year":2003,"finding":"Cripto-1 activates MAPK and AKT pathways independently of Nodal and ALK4 by binding to Glypican-1 and activating c-Src; this ALK4-independent pathway is required for CR-1-induced in vitro transformation and migration.","method":"Co-immunoprecipitation, kinase assays, migration/transformation assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional migration/transformation assays, single lab, two orthogonal methods","pmids":["12649175"],"is_preprint":false},{"year":2004,"finding":"An activin-A M108A mutant retains wild-type affinity for ActRII but cannot form a crosslinked complex with ALK4 in the presence of ActRII, indicating disrupted ALK4 binding; this mutant antagonizes activin-A and myostatin but not TGF-β signaling, confirming it as a type II receptor antagonist that blocks ALK4-dependent ligands.","method":"In vitro crosslinking, receptor-binding assay, cell-based signaling assay, mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro crosslinking with mutagenesis plus functional antagonism assay, single lab, multiple orthogonal methods","pmids":["15123686"],"is_preprint":false},{"year":2004,"finding":"ALK4 co-immunoprecipitates with multiple TGF-β ligands (Activin-Vg1 chimera, Xnr1, derriere, endogenous Vg1); functionally, ALK4 signaling is required for Vg1-mediated LR axis determination but not for Xnr1- or derriere-mediated LR effects, demonstrating ligand-specific utilization of the ALK4 pathway.","method":"Co-immunoprecipitation, dominant-negative receptor injection in Xenopus embryos, morpholino knockdown","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus epistasis via dominant-negative and morpholino, single lab","pmids":["15063168"],"is_preprint":false},{"year":2006,"finding":"Activin B can signal through ALK7 (in addition to ALK4) in immortalized gonadotrope (LβT2) cells to stimulate Fshb transcription; both ALK4(TD) and ALK7(TD) phosphorylate Smad2/3, and the effects of both receptors on Fshb promoter activity require Smad3.","method":"RT-PCR, transfection of constitutively active and kinase-dead receptors, reporter assay, siRNA knockdown, Western blot","journal":"Reproductive biology and endocrinology : RB&E","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple receptor constructs with reporter assay and Smad3 knockdown, single lab","pmids":["17040568"],"is_preprint":false},{"year":2009,"finding":"The Cripto CFC domain interacts with the extracellular domain of ALK4 with a KD in the micromolar range; NMR structural characterization identifies H120 and W124 as externally exposed residues, and molecular docking indicates these and prior mutagenesis-identified residues contribute to the CFC-ALK4 protein-protein interface.","method":"Chemical synthesis of CFC domain, NMR spectroscopy, SPR binding assay, molecular docking","journal":"Journal of peptide science","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR structure plus SPR binding, single lab, no mutagenesis validation in cells","pmids":["19035567"],"is_preprint":false},{"year":2010,"finding":"Conditional keratinocyte-specific knockout of Acvr1b (K14-Cre) causes hair follicle cycling defects and progressive hair loss, as well as persistent epidermal cell proliferation, demonstrating that Acvr1b signaling is required for hair follicle cycling and skin epithelial homeostasis.","method":"Conditional knockout (Cre-lox), histological analysis","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — tissue-specific KO with defined histological phenotype, single lab","pmids":["21191412"],"is_preprint":false},{"year":2013,"finding":"Inhibition of ALK4/ALK7 signaling (but not ALK5 alone) in developing mouse testis impairs male germ cell differentiation and mitotic arrest entry, while ALK4/5/7 inhibition blocks testis cord formation during the sex-determining period; Nodal is specifically expressed in male germ cells and Nanog expression is reduced when ALK4/5/7 signaling is blocked.","method":"Pharmacological inhibition (SB431542), FACS-purified gonadal cell gene expression analysis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition with cell-type-specific gene expression readout, single lab","pmids":["23342175"],"is_preprint":false},{"year":2015,"finding":"Activin A induces human trophoblast invasion by activating ALK4–SMAD2/3–SMAD4 signaling to upregulate SNAIL, which then transcriptionally induces MMP2; siRNA knockdown of ALK4, SMAD2/3, or SMAD4 abolishes activin A-induced SNAIL and MMP2 production and trophoblast invasion.","method":"siRNA knockdown, Western blot, RT-qPCR, Matrigel invasion assay","journal":"The Journal of clinical endocrinology and metabolism","confidence":"High","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown of multiple pathway components with multiple functional readouts in both immortalized and primary cells, single lab","pmids":["26305619"],"is_preprint":false},{"year":2016,"finding":"ALK4 knockdown via antisense oligonucleotides in mdx mice reduces Alk4 expression, inhibits myostatin activity, and increases myoblast differentiation in vitro; paradoxically, in vivo Alk4 inhibition reduces muscle mass (~10%) and increases signs of muscle atrophy, demonstrating ALK4 plays dual roles in muscle atrophy and regeneration.","method":"Antisense oligonucleotide exon skipping, in vitro differentiation assay, in vivo muscle mass measurement, RNAseq","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — AON-mediated knockdown with in vitro and in vivo functional readouts plus transcriptome analysis, single lab","pmids":["27733450"],"is_preprint":false},{"year":2017,"finding":"ALK4 mediates activin A/ALK4/Smad2/3 signaling in atrial fibroblasts; ALK4-deficient mice subjected to angiotensin-II show reduced atrial fibroblast activation, blunted atrial enlargement and fibrosis, and reduced atrial fibrillation vulnerability compared to wild-type littermates.","method":"ALK4 haplodeficiency mouse model, electrophysiological studies, histology, immunostaining","journal":"Basic research in cardiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with defined cardiac phenotype, single lab","pmids":["28639003"],"is_preprint":false},{"year":2019,"finding":"ALK4-mediated Smad3 (but not Smad2) phosphorylation promotes cadmium-induced cell death in renal proximal tubular HK-2 cells via Akt signaling; siRNA knockdown of ALK4 or Smad3, or treatment with SIS3 (Smad3 inhibitor), suppresses cadmium-induced cell death. Separately, ALK4/5 blockade protects against erastin-induced ferroptosis by hyperactivating Nrf2 signaling.","method":"siRNA knockdown, pharmacological inhibition, Western blot, cell viability assay","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown plus pharmacological inhibition with specific pathway readouts, single lab","pmids":["30804470"],"is_preprint":false},{"year":2019,"finding":"ALK4-SMAD2/3-SMAD4 signaling mediates activin A-induced suppression of PTX3 expression in human granulosa-lutein cells; phosphorylated SMADs bind directly to the PTX3 promoter as demonstrated by ChIP analysis.","method":"siRNA knockdown, Western blot, RT-qPCR, ChIP assay","journal":"Molecular and cellular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown of multiple pathway components plus ChIP promoter binding, single lab","pmids":["31185247"],"is_preprint":false},{"year":2020,"finding":"ALK4 signaling in medium spiny neurons (MSNs) of the nucleus accumbens induces SMAD2 interaction with SATB1, promoting SATB1 nuclear translocation and repositioning within the somatostatin gene promoter; ALK4 loss-of-function in MGE GABAergic neurons reduces specific somatostatin interneuron subpopulations in the cortex.","method":"Conditional knockout, co-immunoprecipitation, nuclear fractionation, immunostaining","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with mechanistic Co-IP and nuclear translocation assays, single lab","pmids":["31676717"],"is_preprint":false},{"year":2020,"finding":"Activin A increases trophoblast invasion by upregulating integrin β1 expression through ALK4-activated SMAD2/3-SMAD4 pathway; siRNA knockdown of ALK4 or SMAD4 abolishes activin A-induced integrin β1 upregulation and the increase in cell invasion.","method":"siRNA knockdown, Western blot, RT-qPCR, Matrigel invasion assay","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown of multiple pathway components with invasion assay, single lab","pmids":["33230889"],"is_preprint":false},{"year":2020,"finding":"Activin A induces neuronal differentiation and survival in SK-N-SH neuroblastoma cells via ALK4, but in a SMAD-independent manner: activin A does not induce SMAD2/3 phosphorylation, SMAD2/3-SMAD4 interaction, nuclear SMAD accumulation, or SMAD DNA binding in this context, yet still transactivates TGF-β target genes through ALK4.","method":"Western blot, co-immunoprecipitation, nuclear fractionation, ChIP, siRNA knockdown, neurite outgrowth assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal assays demonstrating SMAD-independent ALK4 signaling, single lab","pmids":["20226172"],"is_preprint":false},{"year":2020,"finding":"Activin A promotes remyelination after ischemic stroke through oligodendroglial ACVR1B; AAV-based ACVR1B shRNA with Olig2 promoter reverses activin A-induced increases in oligodendrocyte number, LFB staining intensity, myelin proteins (MAG, MOG, MBP), and neurological function recovery.","method":"AAV-shRNA knockdown, immunostaining, Western blot, behavioral assays in MCAO/R mouse model","journal":"Experimental neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-type-specific in vivo knockdown with multiple functional and molecular readouts, single lab","pmids":["33345977"],"is_preprint":false},{"year":2021,"finding":"ActRIIB:ALK4-Fc, a heterodimeric fusion protein combining extracellular domains of ALK4 and ActRIIB, has a distinct ligand-binding profile from homodimeric ActRIIB-Fc (by SPR): it sequesters ActRIIB ligands that inhibit muscle growth but does not trap BMP9; it improves muscle mass, function, and NMJ abnormalities in murine DMD, ALS, and disuse atrophy models.","method":"Surface plasmon resonance, in vivo mouse models (DMD, ALS, disuse atrophy), muscle function assays, histology","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — SPR binding characterization plus multiple in vivo models with functional readouts, single lab with multiple orthogonal methods","pmids":["33586684"],"is_preprint":false},{"year":2022,"finding":"Dopamine D1 receptor signaling synergizes with activin/ALK4/Smad3 signaling to potentiate ΔFosB mRNA generation in nucleus accumbens MSNs via activation of RNA-binding protein PCBP1; concurrent D1 and ALK4 activation induces PCBP1-Smad3 interaction, nuclear translocation, and binding to FosB exon-4/intron-4. ALK4 ablation in MSNs impairs ΔFosB induction and behavioral sensitization to cocaine.","method":"Conditional knockout, co-immunoprecipitation, nuclear fractionation, RIP (RNA immunoprecipitation), behavioral sensitization assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional KO plus multiple biochemical assays (Co-IP, nuclear fractionation, RIP) and behavioral phenotype, single lab","pmids":["35730718"],"is_preprint":false},{"year":2022,"finding":"Simultaneous muscle-specific double knockout of Tgfbr1 and Acvr1b in mice induces substantial skeletal muscle hypertrophy (single knockouts do not), mediated by increased Akt/p70S6K phosphorylation and reduced E3 ligase expression; double KO also increases satellite cell and macrophage numbers and improves post-injury regeneration.","method":"Double conditional knockout, phospho-Western blot, histology, cardiotoxin injury model","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis via double KO with multiple orthogonal molecular and cellular readouts, rigorous single study","pmids":["35323108"],"is_preprint":false},{"year":2022,"finding":"ALK4 is preferentially expressed in adipocyte precursors where it suppresses differentiation by inhibiting CEBPα and PPARγ expression; ALK4 deletion induces premature adipocyte differentiation rescued by CEBPα knockdown, establishing that ALK4 promotes adipose tissue hyperplasia by restraining precursor differentiation.","method":"Conditional knockout, siRNA knockdown, gene expression analysis, adipocyte differentiation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with rescue experiment (CEBPα knockdown), single lab","pmids":["36403856"],"is_preprint":false},{"year":2022,"finding":"Activin A modulates betaglycan shedding via the ALK4-SMAD3 (but not SMAD2)-dependent pathway in endometriotic cells; activin A stimulation reduces soluble betaglycan release in an ALK4/SMAD3-dependent manner confirmed by siRNA knockdown and specific inhibitors.","method":"siRNA knockdown, pharmacological inhibition (LY364947/SIS3), ELISA, Western blot","journal":"Biomolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — siRNA knockdown plus pharmacological inhibition identifying ALK4-SMAD3 specificity, single lab","pmids":["36551177"],"is_preprint":false},{"year":2022,"finding":"Activin A promotes trophoblast invasion by upregulating integrin β3 via ALK4-SMAD4 signaling; siRNA knockdown of ALK4 or SMAD4 abolishes activin A-upregulated integrin β3 expression in both immortalized and primary EVT cells.","method":"siRNA knockdown, Western blot, RT-qPCR, invasion assay","journal":"Placenta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown of multiple pathway components in two cell systems with functional invasion readout, single lab","pmids":["36244196"],"is_preprint":false},{"year":2024,"finding":"Loss of Acvr1b (in the presence of Kras oncogene) promotes development of pancreatic intraepithelial neoplasia and IPMN-like precancerous lesions from both acinar and ductal cells, and acute pancreatitis accelerates precancerous lesion development specifically when acinar cells carry both mutations.","method":"Conditional knockout (Ptf1aCreER and Sox9CreER), MRI, histopathology, immunohistochemistry","journal":"Cellular and molecular gastroenterology and hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two cell-type-specific conditional KO models with defined histological phenotypes, single lab","pmids":["39111635"],"is_preprint":false},{"year":2025,"finding":"Cripto-1 acts as a molecular bridge linking Nodal to ALK4: Nodal, bound to type II receptor ActRIIB, uses Cripto-1 (via its EGF-like domain for Nodal binding and CFC domain for ALK4 binding) to recruit ALK4 and form a ternary complex for SMAD2/3 activation; this contrasts with canonical TGF-β ligand-bridging of type I and II receptors.","method":"AlphaFold3 modeling, surface plasmon resonance, domain-specific antibodies, functional SMAD2/3 signaling assay in NTERA-2 cells","journal":"Protein science : a publication of the Protein Society","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — SPR binding plus functional assay with structural modeling, single lab, no mutagenesis validation","pmids":["39840816"],"is_preprint":false},{"year":2025,"finding":"ALK4 loss promotes cancer progression (enhanced anchorage-independent growth, migration, invasion, EMT) in breast and pancreatic cancer models by increasing N-linked glycosylation of TGF-β receptors via MGAT5 upregulation; galectin-3 binds MGAT5-modified glycoproteins to stabilize surface TGF-β receptors, enhancing canonical TGF-β signaling. Depleting MGAT5 or inhibiting N-glycosylation suppresses ALK4-loss-induced TGF-β signaling.","method":"In vitro KO/knockdown, in vivo cancer models, glycosylation analysis, MGAT5/galectin-3 siRNA knockdown, N-glycosylation inhibition","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cancer models in vitro and in vivo with mechanistic rescue experiments, single lab","pmids":["41408046"],"is_preprint":false},{"year":2025,"finding":"ALK4 deletion in osteoblast progenitors (Alk4 cKO mice) increases trabecular bone acquisition, osteoblast number, and bone formation rate; mice are protected against early age-related trabecular bone loss, and an Alk4-Fc ligand trap prevents bone loss in aged mice, indicating Activin/ALK4 signaling suppresses osteoblast activity.","method":"Conditional knockout, bone histomorphometry, ligand trap injection in aged mice, micro-CT","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO and pharmacological ligand-trap approach with bone phenotype readouts, preprint, single lab","pmids":["41278748"],"is_preprint":true},{"year":2025,"finding":"Activin A activates ALK4 in Th17 cells and pulmonary microvascular endothelial cells (PMECs); ALK4 overexpression in Th17 cells increases IL-6 and CTGF in co-cultured PMECs and induces EndoMT; ALK4 activates pSmad2 and pSTAT3 to drive CTGF transcription, and ALK4 inhibitor vactosertib (TEW-7197) suppresses this pathway.","method":"ALK4 overexpression, co-culture, mouse model, ALK4 inhibitor, Western blot, ELISA","journal":"Arthritis & rheumatology (Hoboken, N.J.)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — overexpression and pharmacological inhibition, single lab, no reciprocal binding confirmation","pmids":["40395196"],"is_preprint":false},{"year":2016,"finding":"Activin A stimulates aromatase (P450arom) expression and estradiol secretion in endometrial stromal cells from endometriosis patients via the ALK4-Smad pathway; effects are partially abrogated by an ALK4 inhibitor and Smad4-siRNA knockdown.","method":"Pharmacological inhibition (ALK4 inhibitor), siRNA knockdown, RT-qPCR, Western blot","journal":"BioMed research international","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pharmacological inhibition and single siRNA knockdown, single lab, partial abrogation","pmids":["27833918"],"is_preprint":false},{"year":2011,"finding":"Activin A enhances BAFF expression in mouse macrophages and dendritic cells via ALK4-Smad3 pathway; ALK4 kinase inhibitor SB431542 and dominant-negative Smad3 both abolish activin A-induced BAFF transcription.","method":"RT-PCR, ELISA, pharmacological inhibition (SB431542), dominant-negative Smad3 transfection","journal":"Immune network","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pharmacological inhibition plus DN-Smad3 transfection, single lab, single study","pmids":["22039367"],"is_preprint":false},{"year":2020,"finding":"ALK4-SMAD3-SMAD4 (but not SMAD2 alone for all effects) mediates activin A-induced upregulation of PAI-1 in human granulosa-lutein cells, as demonstrated by siRNA knockdown of ALK4, SMAD3, and SMAD4 each individually abolishing the effect.","method":"siRNA knockdown, Western blot, RT-qPCR, ELISA","journal":"Molecular and cellular endocrinology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — siRNA knockdown with single endpoint, single lab","pmids":["31982478"],"is_preprint":false},{"year":2021,"finding":"Activin A inhibits ox-LDL-induced foam cell formation and upregulates ABCA1/ABCG1 in RAW 264.7 macrophages via Alk4-Smad2 signaling; SB-431542 abolishes these effects and Smad2 knockdown reverses inhibition of ox-LDL uptake, while Smad3 or Smad4 knockdown does not affect uptake but does reverse ABCA1/ABCG1 upregulation.","method":"SB-431542 pharmacological inhibition, siRNA knockdown, Western blot, RT-qPCR, confocal microscopy","journal":"Steroids","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pharmacological inhibition plus siRNA knockdown, single lab, single study","pmids":["34237315"],"is_preprint":false},{"year":2025,"finding":"GDF-9-overexpressing extracellular vesicles activate ACVR1B, which induces SMAD2 phosphorylation and nuclear translocation in granulosa cells to rescue ovarian function in aging mice.","method":"siRNA/genetic modification of EVs, Western blot for SMAD2 phosphorylation and nuclear fractionation, in vivo ovarian function assays","journal":"Science advances","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect evidence via EV delivery with limited mechanistic detail in abstract","pmids":["41370396"],"is_preprint":false}],"current_model":"ACVR1B (ALK4) is a type I transmembrane serine/threonine kinase receptor that forms heteromeric complexes with type II activin receptors (ActRII/IIB) upon binding of activins, Nodal (in the presence of the co-receptor Cripto-1), and related TGF-β superfamily ligands, leading to phosphorylation of SMAD2/3 and downstream transcriptional responses; its extracellular domain contains a defined hydrophobic binding surface for activin-A (Leu40, Ile70, Val73, Leu75, Pro77), a kinase domain loop (between subdomains IV–V) that confers signaling specificity for dorsal mesodermal induction, and it can also signal in a SMAD-independent manner in specific cellular contexts; functionally, ALK4 is required for gastrulation and left-right axis determination in vertebrate embryos, regulates hair follicle cycling, somatostatin interneuron specification, skeletal muscle atrophy/regeneration balance, adipocyte precursor differentiation, atrial fibrosis, and trophoblast invasion, and acts as a tumor suppressor in pancreatic cancer partly by restraining canonical TGF-β signaling through a mechanism involving control of TGF-β receptor glycosylation."},"narrative":{"mechanistic_narrative":"ACVR1B (ALK4) is a type I activin receptor that transduces signals from activins, Nodal, and related TGF-β superfamily ligands by forming heteromeric complexes with type II activin receptors and phosphorylating SMAD2/3 to drive transcriptional responses governing embryonic patterning and tissue homeostasis [PMID:9512518, PMID:9367435, PMID:26305619]. Ligand engagement occurs through a defined hydrophobic surface on the ALK4 extracellular domain (Leu40, Ile70, Val73, Leu75, Pro77) that binds activin-A in the presence of a type II receptor [PMID:12665502, PMID:15123686], while Nodal signaling additionally requires the co-receptor Cripto-1, which bridges Nodal (bound to ActRIIB) to ALK4 via its EGF-like and CFC domains to assemble a SMAD2/3-activating ternary complex [PMID:11485994, PMID:11909953, PMID:39840816]. Signaling specificity is encoded both in a kinase-subdomain IV–V loop that confers dorsal mesoderm-inducing capacity [PMID:9367435, PMID:10075688] and in selective downstream use of SMAD2 versus SMAD3, with SMAD3-biased output controlling distinct gene programs and, in some contexts, SMAD-independent signaling occurring through ALK4 [PMID:26305619, PMID:20226172]. Across tissues, ALK4 sets the balance between proliferation/differentiation and quiescence: it is required for gastrulation and left-right axis determination [PMID:9512518, PMID:15063168], restrains skeletal muscle growth in cooperation with TGFβRI [PMID:35323108], suppresses adipocyte precursor differentiation via CEBPα/PPARγ [PMID:36403856], drives activin A-induced trophoblast invasion through SNAIL/MMP2 and integrin upregulation [PMID:26305619, PMID:33230889, PMID:36244196], and mediates atrial fibrosis [PMID:28639003]. In the nucleus accumbens, ALK4 signaling directs SMAD2/SATB1 and SMAD3/PCBP1 partnerships controlling somatostatin interneuron specification and cocaine-induced ΔFosB induction [PMID:31676717, PMID:35730718]. ALK4 acts as a tumor suppressor: it is somatically mutated in pancreatic carcinoma [PMID:11248065], and its loss cooperates with Kras to promote pancreatic neoplasia [PMID:39111635] and accelerates breast and pancreatic cancer progression by elevating MGAT5-dependent N-glycosylation that stabilizes surface TGF-β receptors and amplifies canonical TGF-β signaling [PMID:41408046]. The small molecule SB-431542 selectively inhibits ALK4/5/7 kinase activity, a tool widely used to dissect this pathway [PMID:12065756].","teleology":[{"year":1997,"claim":"Established that ALK4 signaling alone can establish threshold mesodermal responses and specify dorsal/anterior fates, distinguishing it from ventralizing ALK2 signaling.","evidence":"Constitutively active receptor mRNA injection and gene expression analysis in Xenopus animal caps","pmids":["9367435"],"confidence":"High","gaps":["Identity of endogenous ligands driving dorsal induction in vivo not resolved","Downstream transcriptional mechanism not defined"]},{"year":1998,"claim":"Demonstrated genetically that ALK4 forms complexes with type II activin receptors and is required for gastrulation and primitive streak formation, anchoring its developmental role.","evidence":"Gene targeting/knockout and chimera analysis in mice","pmids":["9512518"],"confidence":"High","gaps":["Does not distinguish which ligand(s) act through ALK4 at gastrulation","Molecular basis of streak requirement not addressed"]},{"year":1999,"claim":"Mapped signaling specificity to a seven-residue kinase subdomain IV–V loop, showing receptor specificity is encoded intracellularly rather than solely by ligand binding.","evidence":"Chimeric receptor construction and Xenopus animal cap assay","pmids":["10075688"],"confidence":"Medium","gaps":["Structural basis of how the loop dictates SMAD/effector selection unknown","Single lab, single study"]},{"year":2000,"claim":"Defined a disease-relevant dominant-negative mechanism whereby truncated ALK4 isoforms in pituitary tumors sequester type II receptors and block antiproliferative activin signaling.","evidence":"Reciprocal Co-IP, reporter and proliferation assays in transfected cells","pmids":["11117535"],"confidence":"High","gaps":["In vivo contribution of truncated isoforms to tumorigenesis not tested","Generality across tumor types unknown"]},{"year":2001,"claim":"Showed ALK4 mediates Nodal signaling only with the co-receptor Cripto, and is the obligate type I receptor for a broad set of mesoderm-inducing ligands, defining its ligand range.","evidence":"Receptor reconstitution, Co-IP, and dominant-negative epistasis in Xenopus; somatic mutation sequencing in pancreatic cancer","pmids":["11485994","11248065"],"confidence":"High","gaps":["Stoichiometry of the Nodal–Cripto–ALK4 complex not resolved at this stage","Functional consequence of pancreatic somatic mutations on receptor activity not directly tested"]},{"year":2002,"claim":"Established Cripto-1 as a direct ALK4-binding co-receptor that gates Nodal-dependent SMAD2 phosphorylation, while Cripto's MAPK/AKT activation is ALK4-independent.","evidence":"Phage display, Co-IP, FACS, and reporter assays in mammalian epithelial cells","pmids":["11909953"],"confidence":"High","gaps":["Domain-level interface not yet defined","Quantitative affinity not measured"]},{"year":2002,"claim":"Provided a selective pharmacological tool by showing SB-431542 inhibits ALK4/5/7 but not other ALKs or MAPK pathways, enabling specific dissection of SMAD2/3 signaling.","evidence":"In vitro kinase assays and cell-based signaling readouts","pmids":["12065756"],"confidence":"High","gaps":["Cannot distinguish ALK4 from ALK5/ALK7 within the pathway","No structural basis for selectivity provided"]},{"year":2003,"claim":"Defined the activin-A binding surface on ALK4 at the residue level and confirmed binding requires the type II receptor, linking structure to ligand engagement.","evidence":"Site-directed mutagenesis, dominant-negative assays, and 125I-activin-A crosslinking","pmids":["12665502","12649175"],"confidence":"High","gaps":["No co-crystal structure of the ternary ligand–receptor complex","Whether the same surface engages other ligands not tested"]},{"year":2004,"claim":"Confirmed ALK4 as the type I receptor selectively engaged by activin-A and myostatin (but not TGF-β) and demonstrated ligand-specific use of ALK4 in left-right axis determination.","evidence":"Activin-A mutant crosslinking and antagonism assays; Co-IP and dominant-negative/morpholino epistasis in Xenopus","pmids":["15123686","15063168"],"confidence":"High","gaps":["Mechanism that channels Vg1 but not Xnr1/derriere to ALK4 unknown","Type II receptor partner specificity not dissected"]},{"year":2009,"claim":"Characterized the Cripto CFC–ALK4 interface structurally, identifying the micromolar binding mode and key exposed residues.","evidence":"Synthetic CFC domain, NMR, SPR binding, and molecular docking","pmids":["19035567"],"confidence":"Medium","gaps":["No cellular mutagenesis validation of docking-predicted contacts","Affinity is low/micromolar, physiological relevance of isolated domain unclear"]},{"year":2010,"claim":"Demonstrated a tissue-homeostatic role: keratinocyte ALK4 is required for hair follicle cycling and restrains epidermal proliferation.","evidence":"Keratinocyte-specific conditional knockout with histology","pmids":["21191412"],"confidence":"Medium","gaps":["Ligand and SMAD effector driving the phenotype not identified","Molecular target genes not defined"]},{"year":2015,"claim":"Resolved a complete signaling-to-phenotype axis for trophoblast invasion, with ALK4–SMAD2/3–SMAD4 upregulating SNAIL and then MMP2.","evidence":"siRNA knockdown of multiple pathway components, expression analysis, and Matrigel invasion assays","pmids":["26305619"],"confidence":"High","gaps":["In vivo relevance to placentation not tested","Direct SMAD binding to SNAIL promoter not shown here"]},{"year":2017,"claim":"Established ALK4 as a driver of pathological fibrosis, with ALK4-deficient mice protected from atrial fibrosis and arrhythmia.","evidence":"ALK4 haplodeficiency mouse model with electrophysiology and histology","pmids":["28639003"],"confidence":"Medium","gaps":["Cell-autonomous versus paracrine contribution not fully separated","Downstream profibrotic transcriptional targets not defined"]},{"year":2016,"claim":"Revealed dual, context-dependent ALK4 roles in muscle, where knockdown enhances myoblast differentiation in vitro yet promotes atrophy in vivo.","evidence":"Antisense oligonucleotide knockdown with in vitro/in vivo functional readouts and RNAseq in mdx mice","pmids":["27733450"],"confidence":"Medium","gaps":["Reconciliation of opposing in vitro/in vivo effects mechanistically unresolved","Contribution of redundant type I receptors not addressed"]},{"year":2020,"claim":"Defined neuronal ALK4 mechanisms in the nucleus accumbens, showing SMAD2-driven SATB1 nuclear repositioning controls somatostatin interneuron specification.","evidence":"Conditional knockout, Co-IP, nuclear fractionation, and immunostaining","pmids":["31676717"],"confidence":"Medium","gaps":["Upstream ligand in MSNs not defined","Direct SMAD2–SATB1 binding interface not mapped"]},{"year":2020,"claim":"Demonstrated SMAD2/3 effector specificity and SMAD-independent ALK4 output, with distinct SMAD2-, SMAD3-, and SMAD-independent programs across cell types.","evidence":"siRNA knockdown, ChIP, nuclear fractionation, and functional assays in trophoblast, neuroblastoma, granulosa, and renal cells","pmids":["33230889","20226172","31185247","30804470"],"confidence":"Medium","gaps":["Molecular basis selecting SMAD2 vs SMAD3 vs SMAD-independent output unknown","Many contexts rely on a single lab"]},{"year":2022,"claim":"Established ALK4 as a redundant brake on skeletal muscle growth: combined Tgfbr1/Acvr1b deletion triggers hypertrophy via Akt/p70S6K and reduced E3 ligases, while single deletion does not.","evidence":"Muscle-specific double conditional knockout with phospho-Western, histology, and injury models","pmids":["35323108"],"confidence":"High","gaps":["Ligand redundancy upstream not fully resolved","Whether SMAD or non-SMAD signaling enforces the brake not separated"]},{"year":2022,"claim":"Expanded ALK4's homeostatic roles to adipose hyperplasia, with ALK4 suppressing precursor differentiation through CEBPα/PPARγ, and consolidated cocaine-related ΔFosB induction via SMAD3–PCBP1.","evidence":"Conditional knockout with rescue (CEBPα knockdown); conditional KO, Co-IP, RIP, and behavioral sensitization","pmids":["36403856","35730718"],"confidence":"High","gaps":["Ligand driving adipose precursor ALK4 signaling not identified","How D1 signaling converges on ALK4/SMAD3 mechanistically incomplete"]},{"year":2024,"claim":"Established ALK4 as a Kras-cooperating pancreatic tumor suppressor whose loss promotes precancerous lesions from acinar and ductal cells, accelerated by pancreatitis.","evidence":"Cell-type-specific conditional knockouts with MRI and histopathology","pmids":["39111635"],"confidence":"Medium","gaps":["Molecular tumor-suppressive mechanism not defined in this study","Progression to invasive carcinoma not quantified"]},{"year":2025,"claim":"Provided a molecular mechanism for ALK4 tumor suppression: ALK4 loss raises MGAT5-dependent N-glycosylation and galectin-3-mediated stabilization of surface TGF-β receptors, amplifying canonical TGF-β signaling and progression.","evidence":"In vitro KO/knockdown, in vivo cancer models, glycosylation analysis, and MGAT5/galectin-3 rescue experiments","pmids":["41408046"],"confidence":"Medium","gaps":["How ALK4 controls MGAT5 expression not mechanistically resolved","Single lab"]},{"year":2025,"claim":"Refined the Nodal signaling architecture, showing Cripto-1 bridges Nodal–ActRIIB to ALK4 via distinct EGF-like and CFC domains, contrasting with canonical ligand-bridging.","evidence":"AlphaFold3 modeling, SPR, domain-specific antibodies, and SMAD2/3 assays in NTERA-2 cells","pmids":["39840816"],"confidence":"Medium","gaps":["No mutagenesis validation of modeled interfaces","No experimental structure of the ternary complex"]},{"year":null,"claim":"It remains unresolved what determines ALK4's choice among SMAD2, SMAD3, and SMAD-independent outputs across tissues, and how upstream ligand and type II receptor combinations select these programs.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying structural/biochemical model of effector selection","Tissue-specific ligand inputs largely inferred rather than mapped","Cause-effect link between glycosylation control and SMAD output not integrated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,16]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[12,16]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[4,6,8]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[7]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,6,8]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,16]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,1,4]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[5,31,33]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[16,20,26]}],"complexes":["ALK4–type II activin receptor (ActRII/IIB) heteromeric receptor complex","Nodal–Cripto-1–ALK4–ActRIIB ternary signaling complex"],"partners":["ACVR2B","TDGF1","SMAD2","SMAD3","SMAD4","TGFBR1","SATB1","PCBP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P36896","full_name":"Activin receptor type-1B","aliases":["Activin receptor type IB","ACTR-IB","Activin receptor-like kinase 4","ALK-4","Serine/threonine-protein kinase receptor R2","SKR2"],"length_aa":505,"mass_kda":56.8,"function":"Transmembrane serine/threonine kinase activin type-1 receptor forming an activin receptor complex with activin receptor type-2 (ACVR2A or ACVR2B). Transduces the activin signal from the cell surface to the cytoplasm and is thus regulating a many physiological and pathological processes including neuronal differentiation and neuronal survival, hair follicle development and cycling, FSH production by the pituitary gland, wound healing, extracellular matrix production, immunosuppression and carcinogenesis. Activin is also thought to have a paracrine or autocrine role in follicular development in the ovary. Within the receptor complex, type-2 receptors (ACVR2A and/or ACVR2B) act as a primary activin receptors whereas the type-1 receptors like ACVR1B act as downstream transducers of activin signals. Activin binds to type-2 receptor at the plasma membrane and activates its serine-threonine kinase. The activated receptor type-2 then phosphorylates and activates the type-1 receptor such as ACVR1B. Once activated, the type-1 receptor binds and phosphorylates the SMAD proteins SMAD2 and SMAD3, on serine residues of the C-terminal tail. Soon after their association with the activin receptor and subsequent phosphorylation, SMAD2 and SMAD3 are released into the cytoplasm where they interact with the common partner SMAD4. This SMAD complex translocates into the nucleus where it mediates activin-induced transcription. Inhibitory SMAD7, which is recruited to ACVR1B through FKBP1A, can prevent the association of SMAD2 and SMAD3 with the activin receptor complex, thereby blocking the activin signal. Activin signal transduction is also antagonized by the binding to the receptor of inhibin-B via the IGSF1 inhibin coreceptor. ACVR1B also phosphorylates TDP2","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/P36896/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ACVR1B","classification":"Not Classified","n_dependent_lines":22,"n_total_lines":1208,"dependency_fraction":0.018211920529801324},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ACVR1B","total_profiled":1310},"omim":[{"mim_id":"620847","title":"BONE MORPHOGENETIC PROTEIN 8A; BMP8A","url":"https://www.omim.org/entry/620847"},{"mim_id":"619560","title":"MICRO RNA 135B; MIR135B","url":"https://www.omim.org/entry/619560"},{"mim_id":"613992","title":"PROTEIN PHOSPHATASE 2, REGULATORY SUBUNIT B, DELTA; PPP2R2D","url":"https://www.omim.org/entry/613992"},{"mim_id":"613347","title":"PANCREATIC CANCER, SUSCEPTIBILITY TO, 2","url":"https://www.omim.org/entry/613347"},{"mim_id":"609705","title":"MICRO RNA 24-1; MIR24-1","url":"https://www.omim.org/entry/609705"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Vesicles","reliability":"Additional"},{"location":"Primary cilium","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ACVR1B"},"hgnc":{"alias_symbol":["ALK4","SKR2","ActRIB"],"prev_symbol":["ACVRLK4"]},"alphafold":{"accession":"P36896","domains":[{"cath_id":"2.10.60.10","chopping":"31-95","consensus_level":"high","plddt":84.0892,"start":31,"end":95},{"cath_id":"3.30.200.20","chopping":"187-284","consensus_level":"medium","plddt":86.9721,"start":187,"end":284},{"cath_id":"1.10.510.10","chopping":"288-505","consensus_level":"medium","plddt":94.7679,"start":288,"end":505}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P36896","model_url":"https://alphafold.ebi.ac.uk/files/AF-P36896-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P36896-F1-predicted_aligned_error_v6.png","plddt_mean":83.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ACVR1B","jax_strain_url":"https://www.jax.org/strain/search?query=ACVR1B"},"sequence":{"accession":"P36896","fasta_url":"https://rest.uniprot.org/uniprotkb/P36896.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P36896/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P36896"}},"corpus_meta":[{"pmid":"12065756","id":"PMC_12065756","title":"SB-431542 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Th17-Involved Endothelial-Mesenchymal Transition in Systemic Lupus Erythematosus-Associated Pulmonary Arterial Hypertension.","date":"2025","source":"Arthritis & rheumatology (Hoboken, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/40395196","citation_count":1,"is_preprint":false},{"pmid":"41370396","id":"PMC_41370396","title":"Repair of female reproductive function by GDF-9-overexpressing extracellular vesicles via ACVR1B/SMAD2 regulation in ovarian granulosa.","date":"2025","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/41370396","citation_count":1,"is_preprint":false},{"pmid":"37020544","id":"PMC_37020544","title":"Abundance of ACVR1B transcript is elevated during septic conditions: Perspectives obtained from a hands-on reductionist investigation.","date":"2023","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/37020544","citation_count":1,"is_preprint":false},{"pmid":"35920165","id":"PMC_35920165","title":"The activin receptor ligand trap 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Japanese journal of hygiene","url":"https://pubmed.ncbi.nlm.nih.gov/33342936","citation_count":1,"is_preprint":false},{"pmid":"39544188","id":"PMC_39544188","title":"Genetic insights into endurance athlete status: A meta-analysis of ACVR1B, AGT, FTO, IL-6, and NRF2 gene polymorphisms.","date":"2024","source":"Journal of biological methods","url":"https://pubmed.ncbi.nlm.nih.gov/39544188","citation_count":1,"is_preprint":false},{"pmid":"37462480","id":"PMC_37462480","title":"[Up-regulated expression of miR-576 inhibits ALK4 expression, regulates JAK/STAT signaling pathway and promotes proliferation and migration of prostatic cancer cells].","date":"2022","source":"Zhonghua nan ke xue = National journal of andrology","url":"https://pubmed.ncbi.nlm.nih.gov/37462480","citation_count":1,"is_preprint":false},{"pmid":"41408046","id":"PMC_41408046","title":"Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/41408046","citation_count":0,"is_preprint":false},{"pmid":"40494204","id":"PMC_40494204","title":"Activin A exacerbates neonatal necrotizing enterocolitis via ALK4-mediated apoptosis and barrier disruption.","date":"2025","source":"International immunopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40494204","citation_count":0,"is_preprint":false},{"pmid":"41278748","id":"PMC_41278748","title":"Targeting the Alk4 pathway protects against age-related bone loss.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41278748","citation_count":0,"is_preprint":false},{"pmid":"42137245","id":"PMC_42137245","title":"RUNX1-mediated repression of miR-24 promotes hepatic stellate cell activation and liver fibrosis by targeting the ALK4/Smad3 signaling pathway.","date":"2026","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/42137245","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.01.23.25321014","title":"Activin A-activated ALK4 induces pathogenic Th17 involvement in endothelial–mesenchymal transition in systemic lupus erythematosus-associated pulmonary arterial hypertension","date":"2025-01-25","source":"bioRxiv","url":"https://doi.org/10.1101/2025.01.23.25321014","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.02.07.637115","title":"CryoEM structure of ALK2:BMP6 reveals distinct mechanisms that allow ALK2 to interact with both BMP and Activin ligands","date":"2025-02-08","source":"bioRxiv","url":"https://doi.org/10.1101/2025.02.07.637115","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":42458,"output_tokens":9540,"usd":0.135237,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":19743,"output_tokens":6261,"usd":0.12762,"stage2_stop_reason":"end_turn"},"total_usd":0.262857,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"ActRIB (ACVR1B) forms heteromeric complexes with type II activin receptors to mediate activin signaling; genetic knockout in mice shows it is required for egg cylinder organization and gastrulation, with chimera analysis demonstrating a role in primitive streak formation but not mesoderm differentiation per se.\",\n      \"method\": \"Gene targeting/knockout, chimera analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with defined developmental phenotype replicated across multiple chimera experiments in a single rigorous study\",\n      \"pmids\": [\"9512518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Constitutively active ALK4 (ALK-4*) induces dose-dependent mesoderm including dorsal/anterior cell types (goosecoid, Xbra) and endoderm in Xenopus animal caps, whereas ALK-2* induces only ventral mesoderm; threshold responses to activin can be established by ALK4 signaling alone, and ventralizing ALK-2* signals antagonize ALK-4* dorsal signals.\",\n      \"method\": \"Constitutively active receptor mRNA injection, Xenopus animal cap assay, gene expression analysis\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — functional reconstitution with constitutively active and dominant-negative receptors, dose-response analysis, replicated across multiple experimental conditions\",\n      \"pmids\": [\"9367435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The loop between kinase subdomains IV and V of ALK4 (seven amino acids) mediates its strong dorsal gene-inducing signaling specificity; transferring this loop from ALK4* to ALK2* confers dorsal gene induction on ALK2*, identifying this loop as a key determinant of ALK4 signaling specificity.\",\n      \"method\": \"Chimeric receptor construction, mRNA injection, Xenopus animal cap assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — chimeric mutagenesis with functional readout, single lab, single study\",\n      \"pmids\": [\"10075688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Truncated ALK4 isoforms (Alk4-2, 4-3, 4-4) found in human pituitary tumors act as dominant-negative receptors: they co-immunoprecipitate with type II activin receptors but are not phosphorylated, blocking wild-type activin signaling and the antiproliferative effects of activin.\",\n      \"method\": \"Coimmunoprecipitation, reporter assays, stable transfection, cell proliferation assay\",\n      \"journal\": \"Molecular endocrinology (Baltimore, Md.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus functional reporter and proliferation assays, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"11117535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"ALK4 mediates Nodal signaling (Xnr1, mouse Nodal) only in the presence of the co-receptor Cripto; ALK4 can directly interact with Cripto. A dominant-negative ALK4 blocks all mesoderm-inducing ligands (Nodal, Xnr1, Xnr2, Xnr4, Activin), while dominant-negative ALK7 specifically blocks Nodal/Xnr1.\",\n      \"method\": \"Receptor reconstitution, Co-IP, dominant-negative receptor injection in Xenopus embryos\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — biochemical binding plus functional epistasis in multiple ligand/receptor combinations, replicated across Xenopus and mouse contexts\",\n      \"pmids\": [\"11485994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"ACVR1B harbors somatic mutations in pancreatic carcinoma, establishing it as a mutated tumor-suppressor gene in sporadic pancreatic cancer.\",\n      \"method\": \"Direct sequencing of pancreatic cancer samples\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct sequencing across multiple tumor samples, single lab, establishes somatic mutation status\",\n      \"pmids\": [\"11248065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Cripto-1 binds directly to ALK4 on mammalian epithelial cells (identified by phage display and confirmed by co-immunoprecipitation and FACS); Cripto-1 phosphorylates Smad2 only in the presence of both Nodal and ALK4, while Cripto-1-stimulated MAPK and AKT activation is independent of Nodal and ALK4.\",\n      \"method\": \"Phage display library screening, coimmunoprecipitation, FACS, reporter assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding identified by two orthogonal methods (phage display + Co-IP), plus functional Smad2 phosphorylation assay, single lab\",\n      \"pmids\": [\"11909953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"SB-431542 inhibits ALK4, ALK5, and ALK7 kinase activity but has no effect on other ALK family members (ALK1, 2, 3, 6) or on ERK, JNK, or p38 MAP kinase pathways, making it a selective inhibitor of activin/TGF-β/Nodal type I receptor signaling via SMAD2/3.\",\n      \"method\": \"Biochemical kinase inhibition assay, reporter assays, cell signaling analysis\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assays plus multiple cell-based signaling readouts, widely replicated across many labs\",\n      \"pmids\": [\"12065756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Five hydrophobic residues on the ALK4 extracellular domain (Leu40, Ile70, Val73, Leu75, Pro77) constitute a functional binding surface for activin-A; mutation of these residues to alanine substantially disrupts dominant-negative ALK4 activity and reduces 125I-activin-A crosslinking to ALK4 in the presence of ActRII.\",\n      \"method\": \"Site-directed mutagenesis, dominant-negative inhibition assay, 125I-activin crosslinking\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis combined with biochemical binding assay (crosslinking), multiple mutants tested, single lab\",\n      \"pmids\": [\"12665502\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Cripto-1 activates MAPK and AKT pathways independently of Nodal and ALK4 by binding to Glypican-1 and activating c-Src; this ALK4-independent pathway is required for CR-1-induced in vitro transformation and migration.\",\n      \"method\": \"Co-immunoprecipitation, kinase assays, migration/transformation assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional migration/transformation assays, single lab, two orthogonal methods\",\n      \"pmids\": [\"12649175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"An activin-A M108A mutant retains wild-type affinity for ActRII but cannot form a crosslinked complex with ALK4 in the presence of ActRII, indicating disrupted ALK4 binding; this mutant antagonizes activin-A and myostatin but not TGF-β signaling, confirming it as a type II receptor antagonist that blocks ALK4-dependent ligands.\",\n      \"method\": \"In vitro crosslinking, receptor-binding assay, cell-based signaling assay, mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro crosslinking with mutagenesis plus functional antagonism assay, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"15123686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ALK4 co-immunoprecipitates with multiple TGF-β ligands (Activin-Vg1 chimera, Xnr1, derriere, endogenous Vg1); functionally, ALK4 signaling is required for Vg1-mediated LR axis determination but not for Xnr1- or derriere-mediated LR effects, demonstrating ligand-specific utilization of the ALK4 pathway.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative receptor injection in Xenopus embryos, morpholino knockdown\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus epistasis via dominant-negative and morpholino, single lab\",\n      \"pmids\": [\"15063168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Activin B can signal through ALK7 (in addition to ALK4) in immortalized gonadotrope (LβT2) cells to stimulate Fshb transcription; both ALK4(TD) and ALK7(TD) phosphorylate Smad2/3, and the effects of both receptors on Fshb promoter activity require Smad3.\",\n      \"method\": \"RT-PCR, transfection of constitutively active and kinase-dead receptors, reporter assay, siRNA knockdown, Western blot\",\n      \"journal\": \"Reproductive biology and endocrinology : RB&E\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple receptor constructs with reporter assay and Smad3 knockdown, single lab\",\n      \"pmids\": [\"17040568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The Cripto CFC domain interacts with the extracellular domain of ALK4 with a KD in the micromolar range; NMR structural characterization identifies H120 and W124 as externally exposed residues, and molecular docking indicates these and prior mutagenesis-identified residues contribute to the CFC-ALK4 protein-protein interface.\",\n      \"method\": \"Chemical synthesis of CFC domain, NMR spectroscopy, SPR binding assay, molecular docking\",\n      \"journal\": \"Journal of peptide science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR structure plus SPR binding, single lab, no mutagenesis validation in cells\",\n      \"pmids\": [\"19035567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Conditional keratinocyte-specific knockout of Acvr1b (K14-Cre) causes hair follicle cycling defects and progressive hair loss, as well as persistent epidermal cell proliferation, demonstrating that Acvr1b signaling is required for hair follicle cycling and skin epithelial homeostasis.\",\n      \"method\": \"Conditional knockout (Cre-lox), histological analysis\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tissue-specific KO with defined histological phenotype, single lab\",\n      \"pmids\": [\"21191412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Inhibition of ALK4/ALK7 signaling (but not ALK5 alone) in developing mouse testis impairs male germ cell differentiation and mitotic arrest entry, while ALK4/5/7 inhibition blocks testis cord formation during the sex-determining period; Nodal is specifically expressed in male germ cells and Nanog expression is reduced when ALK4/5/7 signaling is blocked.\",\n      \"method\": \"Pharmacological inhibition (SB431542), FACS-purified gonadal cell gene expression analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition with cell-type-specific gene expression readout, single lab\",\n      \"pmids\": [\"23342175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Activin A induces human trophoblast invasion by activating ALK4–SMAD2/3–SMAD4 signaling to upregulate SNAIL, which then transcriptionally induces MMP2; siRNA knockdown of ALK4, SMAD2/3, or SMAD4 abolishes activin A-induced SNAIL and MMP2 production and trophoblast invasion.\",\n      \"method\": \"siRNA knockdown, Western blot, RT-qPCR, Matrigel invasion assay\",\n      \"journal\": \"The Journal of clinical endocrinology and metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown of multiple pathway components with multiple functional readouts in both immortalized and primary cells, single lab\",\n      \"pmids\": [\"26305619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ALK4 knockdown via antisense oligonucleotides in mdx mice reduces Alk4 expression, inhibits myostatin activity, and increases myoblast differentiation in vitro; paradoxically, in vivo Alk4 inhibition reduces muscle mass (~10%) and increases signs of muscle atrophy, demonstrating ALK4 plays dual roles in muscle atrophy and regeneration.\",\n      \"method\": \"Antisense oligonucleotide exon skipping, in vitro differentiation assay, in vivo muscle mass measurement, RNAseq\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — AON-mediated knockdown with in vitro and in vivo functional readouts plus transcriptome analysis, single lab\",\n      \"pmids\": [\"27733450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ALK4 mediates activin A/ALK4/Smad2/3 signaling in atrial fibroblasts; ALK4-deficient mice subjected to angiotensin-II show reduced atrial fibroblast activation, blunted atrial enlargement and fibrosis, and reduced atrial fibrillation vulnerability compared to wild-type littermates.\",\n      \"method\": \"ALK4 haplodeficiency mouse model, electrophysiological studies, histology, immunostaining\",\n      \"journal\": \"Basic research in cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with defined cardiac phenotype, single lab\",\n      \"pmids\": [\"28639003\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ALK4-mediated Smad3 (but not Smad2) phosphorylation promotes cadmium-induced cell death in renal proximal tubular HK-2 cells via Akt signaling; siRNA knockdown of ALK4 or Smad3, or treatment with SIS3 (Smad3 inhibitor), suppresses cadmium-induced cell death. Separately, ALK4/5 blockade protects against erastin-induced ferroptosis by hyperactivating Nrf2 signaling.\",\n      \"method\": \"siRNA knockdown, pharmacological inhibition, Western blot, cell viability assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown plus pharmacological inhibition with specific pathway readouts, single lab\",\n      \"pmids\": [\"30804470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ALK4-SMAD2/3-SMAD4 signaling mediates activin A-induced suppression of PTX3 expression in human granulosa-lutein cells; phosphorylated SMADs bind directly to the PTX3 promoter as demonstrated by ChIP analysis.\",\n      \"method\": \"siRNA knockdown, Western blot, RT-qPCR, ChIP assay\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown of multiple pathway components plus ChIP promoter binding, single lab\",\n      \"pmids\": [\"31185247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALK4 signaling in medium spiny neurons (MSNs) of the nucleus accumbens induces SMAD2 interaction with SATB1, promoting SATB1 nuclear translocation and repositioning within the somatostatin gene promoter; ALK4 loss-of-function in MGE GABAergic neurons reduces specific somatostatin interneuron subpopulations in the cortex.\",\n      \"method\": \"Conditional knockout, co-immunoprecipitation, nuclear fractionation, immunostaining\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with mechanistic Co-IP and nuclear translocation assays, single lab\",\n      \"pmids\": [\"31676717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Activin A increases trophoblast invasion by upregulating integrin β1 expression through ALK4-activated SMAD2/3-SMAD4 pathway; siRNA knockdown of ALK4 or SMAD4 abolishes activin A-induced integrin β1 upregulation and the increase in cell invasion.\",\n      \"method\": \"siRNA knockdown, Western blot, RT-qPCR, Matrigel invasion assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown of multiple pathway components with invasion assay, single lab\",\n      \"pmids\": [\"33230889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Activin A induces neuronal differentiation and survival in SK-N-SH neuroblastoma cells via ALK4, but in a SMAD-independent manner: activin A does not induce SMAD2/3 phosphorylation, SMAD2/3-SMAD4 interaction, nuclear SMAD accumulation, or SMAD DNA binding in this context, yet still transactivates TGF-β target genes through ALK4.\",\n      \"method\": \"Western blot, co-immunoprecipitation, nuclear fractionation, ChIP, siRNA knockdown, neurite outgrowth assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal assays demonstrating SMAD-independent ALK4 signaling, single lab\",\n      \"pmids\": [\"20226172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Activin A promotes remyelination after ischemic stroke through oligodendroglial ACVR1B; AAV-based ACVR1B shRNA with Olig2 promoter reverses activin A-induced increases in oligodendrocyte number, LFB staining intensity, myelin proteins (MAG, MOG, MBP), and neurological function recovery.\",\n      \"method\": \"AAV-shRNA knockdown, immunostaining, Western blot, behavioral assays in MCAO/R mouse model\",\n      \"journal\": \"Experimental neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific in vivo knockdown with multiple functional and molecular readouts, single lab\",\n      \"pmids\": [\"33345977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ActRIIB:ALK4-Fc, a heterodimeric fusion protein combining extracellular domains of ALK4 and ActRIIB, has a distinct ligand-binding profile from homodimeric ActRIIB-Fc (by SPR): it sequesters ActRIIB ligands that inhibit muscle growth but does not trap BMP9; it improves muscle mass, function, and NMJ abnormalities in murine DMD, ALS, and disuse atrophy models.\",\n      \"method\": \"Surface plasmon resonance, in vivo mouse models (DMD, ALS, disuse atrophy), muscle function assays, histology\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — SPR binding characterization plus multiple in vivo models with functional readouts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"33586684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Dopamine D1 receptor signaling synergizes with activin/ALK4/Smad3 signaling to potentiate ΔFosB mRNA generation in nucleus accumbens MSNs via activation of RNA-binding protein PCBP1; concurrent D1 and ALK4 activation induces PCBP1-Smad3 interaction, nuclear translocation, and binding to FosB exon-4/intron-4. ALK4 ablation in MSNs impairs ΔFosB induction and behavioral sensitization to cocaine.\",\n      \"method\": \"Conditional knockout, co-immunoprecipitation, nuclear fractionation, RIP (RNA immunoprecipitation), behavioral sensitization assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO plus multiple biochemical assays (Co-IP, nuclear fractionation, RIP) and behavioral phenotype, single lab\",\n      \"pmids\": [\"35730718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Simultaneous muscle-specific double knockout of Tgfbr1 and Acvr1b in mice induces substantial skeletal muscle hypertrophy (single knockouts do not), mediated by increased Akt/p70S6K phosphorylation and reduced E3 ligase expression; double KO also increases satellite cell and macrophage numbers and improves post-injury regeneration.\",\n      \"method\": \"Double conditional knockout, phospho-Western blot, histology, cardiotoxin injury model\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis via double KO with multiple orthogonal molecular and cellular readouts, rigorous single study\",\n      \"pmids\": [\"35323108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALK4 is preferentially expressed in adipocyte precursors where it suppresses differentiation by inhibiting CEBPα and PPARγ expression; ALK4 deletion induces premature adipocyte differentiation rescued by CEBPα knockdown, establishing that ALK4 promotes adipose tissue hyperplasia by restraining precursor differentiation.\",\n      \"method\": \"Conditional knockout, siRNA knockdown, gene expression analysis, adipocyte differentiation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with rescue experiment (CEBPα knockdown), single lab\",\n      \"pmids\": [\"36403856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Activin A modulates betaglycan shedding via the ALK4-SMAD3 (but not SMAD2)-dependent pathway in endometriotic cells; activin A stimulation reduces soluble betaglycan release in an ALK4/SMAD3-dependent manner confirmed by siRNA knockdown and specific inhibitors.\",\n      \"method\": \"siRNA knockdown, pharmacological inhibition (LY364947/SIS3), ELISA, Western blot\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — siRNA knockdown plus pharmacological inhibition identifying ALK4-SMAD3 specificity, single lab\",\n      \"pmids\": [\"36551177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Activin A promotes trophoblast invasion by upregulating integrin β3 via ALK4-SMAD4 signaling; siRNA knockdown of ALK4 or SMAD4 abolishes activin A-upregulated integrin β3 expression in both immortalized and primary EVT cells.\",\n      \"method\": \"siRNA knockdown, Western blot, RT-qPCR, invasion assay\",\n      \"journal\": \"Placenta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown of multiple pathway components in two cell systems with functional invasion readout, single lab\",\n      \"pmids\": [\"36244196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Loss of Acvr1b (in the presence of Kras oncogene) promotes development of pancreatic intraepithelial neoplasia and IPMN-like precancerous lesions from both acinar and ductal cells, and acute pancreatitis accelerates precancerous lesion development specifically when acinar cells carry both mutations.\",\n      \"method\": \"Conditional knockout (Ptf1aCreER and Sox9CreER), MRI, histopathology, immunohistochemistry\",\n      \"journal\": \"Cellular and molecular gastroenterology and hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two cell-type-specific conditional KO models with defined histological phenotypes, single lab\",\n      \"pmids\": [\"39111635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cripto-1 acts as a molecular bridge linking Nodal to ALK4: Nodal, bound to type II receptor ActRIIB, uses Cripto-1 (via its EGF-like domain for Nodal binding and CFC domain for ALK4 binding) to recruit ALK4 and form a ternary complex for SMAD2/3 activation; this contrasts with canonical TGF-β ligand-bridging of type I and II receptors.\",\n      \"method\": \"AlphaFold3 modeling, surface plasmon resonance, domain-specific antibodies, functional SMAD2/3 signaling assay in NTERA-2 cells\",\n      \"journal\": \"Protein science : a publication of the Protein Society\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — SPR binding plus functional assay with structural modeling, single lab, no mutagenesis validation\",\n      \"pmids\": [\"39840816\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALK4 loss promotes cancer progression (enhanced anchorage-independent growth, migration, invasion, EMT) in breast and pancreatic cancer models by increasing N-linked glycosylation of TGF-β receptors via MGAT5 upregulation; galectin-3 binds MGAT5-modified glycoproteins to stabilize surface TGF-β receptors, enhancing canonical TGF-β signaling. Depleting MGAT5 or inhibiting N-glycosylation suppresses ALK4-loss-induced TGF-β signaling.\",\n      \"method\": \"In vitro KO/knockdown, in vivo cancer models, glycosylation analysis, MGAT5/galectin-3 siRNA knockdown, N-glycosylation inhibition\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cancer models in vitro and in vivo with mechanistic rescue experiments, single lab\",\n      \"pmids\": [\"41408046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALK4 deletion in osteoblast progenitors (Alk4 cKO mice) increases trabecular bone acquisition, osteoblast number, and bone formation rate; mice are protected against early age-related trabecular bone loss, and an Alk4-Fc ligand trap prevents bone loss in aged mice, indicating Activin/ALK4 signaling suppresses osteoblast activity.\",\n      \"method\": \"Conditional knockout, bone histomorphometry, ligand trap injection in aged mice, micro-CT\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO and pharmacological ligand-trap approach with bone phenotype readouts, preprint, single lab\",\n      \"pmids\": [\"41278748\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Activin A activates ALK4 in Th17 cells and pulmonary microvascular endothelial cells (PMECs); ALK4 overexpression in Th17 cells increases IL-6 and CTGF in co-cultured PMECs and induces EndoMT; ALK4 activates pSmad2 and pSTAT3 to drive CTGF transcription, and ALK4 inhibitor vactosertib (TEW-7197) suppresses this pathway.\",\n      \"method\": \"ALK4 overexpression, co-culture, mouse model, ALK4 inhibitor, Western blot, ELISA\",\n      \"journal\": \"Arthritis & rheumatology (Hoboken, N.J.)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — overexpression and pharmacological inhibition, single lab, no reciprocal binding confirmation\",\n      \"pmids\": [\"40395196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Activin A stimulates aromatase (P450arom) expression and estradiol secretion in endometrial stromal cells from endometriosis patients via the ALK4-Smad pathway; effects are partially abrogated by an ALK4 inhibitor and Smad4-siRNA knockdown.\",\n      \"method\": \"Pharmacological inhibition (ALK4 inhibitor), siRNA knockdown, RT-qPCR, Western blot\",\n      \"journal\": \"BioMed research international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pharmacological inhibition and single siRNA knockdown, single lab, partial abrogation\",\n      \"pmids\": [\"27833918\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Activin A enhances BAFF expression in mouse macrophages and dendritic cells via ALK4-Smad3 pathway; ALK4 kinase inhibitor SB431542 and dominant-negative Smad3 both abolish activin A-induced BAFF transcription.\",\n      \"method\": \"RT-PCR, ELISA, pharmacological inhibition (SB431542), dominant-negative Smad3 transfection\",\n      \"journal\": \"Immune network\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pharmacological inhibition plus DN-Smad3 transfection, single lab, single study\",\n      \"pmids\": [\"22039367\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALK4-SMAD3-SMAD4 (but not SMAD2 alone for all effects) mediates activin A-induced upregulation of PAI-1 in human granulosa-lutein cells, as demonstrated by siRNA knockdown of ALK4, SMAD3, and SMAD4 each individually abolishing the effect.\",\n      \"method\": \"siRNA knockdown, Western blot, RT-qPCR, ELISA\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — siRNA knockdown with single endpoint, single lab\",\n      \"pmids\": [\"31982478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Activin A inhibits ox-LDL-induced foam cell formation and upregulates ABCA1/ABCG1 in RAW 264.7 macrophages via Alk4-Smad2 signaling; SB-431542 abolishes these effects and Smad2 knockdown reverses inhibition of ox-LDL uptake, while Smad3 or Smad4 knockdown does not affect uptake but does reverse ABCA1/ABCG1 upregulation.\",\n      \"method\": \"SB-431542 pharmacological inhibition, siRNA knockdown, Western blot, RT-qPCR, confocal microscopy\",\n      \"journal\": \"Steroids\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pharmacological inhibition plus siRNA knockdown, single lab, single study\",\n      \"pmids\": [\"34237315\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GDF-9-overexpressing extracellular vesicles activate ACVR1B, which induces SMAD2 phosphorylation and nuclear translocation in granulosa cells to rescue ovarian function in aging mice.\",\n      \"method\": \"siRNA/genetic modification of EVs, Western blot for SMAD2 phosphorylation and nuclear fractionation, in vivo ovarian function assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect evidence via EV delivery with limited mechanistic detail in abstract\",\n      \"pmids\": [\"41370396\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ACVR1B (ALK4) is a type I transmembrane serine/threonine kinase receptor that forms heteromeric complexes with type II activin receptors (ActRII/IIB) upon binding of activins, Nodal (in the presence of the co-receptor Cripto-1), and related TGF-β superfamily ligands, leading to phosphorylation of SMAD2/3 and downstream transcriptional responses; its extracellular domain contains a defined hydrophobic binding surface for activin-A (Leu40, Ile70, Val73, Leu75, Pro77), a kinase domain loop (between subdomains IV–V) that confers signaling specificity for dorsal mesodermal induction, and it can also signal in a SMAD-independent manner in specific cellular contexts; functionally, ALK4 is required for gastrulation and left-right axis determination in vertebrate embryos, regulates hair follicle cycling, somatostatin interneuron specification, skeletal muscle atrophy/regeneration balance, adipocyte precursor differentiation, atrial fibrosis, and trophoblast invasion, and acts as a tumor suppressor in pancreatic cancer partly by restraining canonical TGF-β signaling through a mechanism involving control of TGF-β receptor glycosylation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ACVR1B (ALK4) is a type I activin receptor that transduces signals from activins, Nodal, and related TGF-\\u03b2 superfamily ligands by forming heteromeric complexes with type II activin receptors and phosphorylating SMAD2/3 to drive transcriptional responses governing embryonic patterning and tissue homeostasis [#0, #1, #16]. Ligand engagement occurs through a defined hydrophobic surface on the ALK4 extracellular domain (Leu40, Ile70, Val73, Leu75, Pro77) that binds activin-A in the presence of a type II receptor [#8, #10], while Nodal signaling additionally requires the co-receptor Cripto-1, which bridges Nodal (bound to ActRIIB) to ALK4 via its EGF-like and CFC domains to assemble a SMAD2/3-activating ternary complex [#4, #6, #32]. Signaling specificity is encoded both in a kinase-subdomain IV\\u2013V loop that confers dorsal mesoderm-inducing capacity [#1, #2] and in selective downstream use of SMAD2 versus SMAD3, with SMAD3-biased output controlling distinct gene programs and, in some contexts, SMAD-independent signaling occurring through ALK4 [#16, #23]. Across tissues, ALK4 sets the balance between proliferation/differentiation and quiescence: it is required for gastrulation and left-right axis determination [#0, #11], restrains skeletal muscle growth in cooperation with TGF\\u03b2RI [#27], suppresses adipocyte precursor differentiation via CEBP\\u03b1/PPAR\\u03b3 [#28], drives activin A-induced trophoblast invasion through SNAIL/MMP2 and integrin upregulation [#16, #22, #30], and mediates atrial fibrosis [#18]. In the nucleus accumbens, ALK4 signaling directs SMAD2/SATB1 and SMAD3/PCBP1 partnerships controlling somatostatin interneuron specification and cocaine-induced \\u0394FosB induction [#21, #26]. ALK4 acts as a tumor suppressor: it is somatically mutated in pancreatic carcinoma [#5], and its loss cooperates with Kras to promote pancreatic neoplasia [#31] and accelerates breast and pancreatic cancer progression by elevating MGAT5-dependent N-glycosylation that stabilizes surface TGF-\\u03b2 receptors and amplifies canonical TGF-\\u03b2 signaling [#33]. The small molecule SB-431542 selectively inhibits ALK4/5/7 kinase activity, a tool widely used to dissect this pathway [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established that ALK4 signaling alone can establish threshold mesodermal responses and specify dorsal/anterior fates, distinguishing it from ventralizing ALK2 signaling.\",\n      \"evidence\": \"Constitutively active receptor mRNA injection and gene expression analysis in Xenopus animal caps\",\n      \"pmids\": [\"9367435\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of endogenous ligands driving dorsal induction in vivo not resolved\", \"Downstream transcriptional mechanism not defined\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Demonstrated genetically that ALK4 forms complexes with type II activin receptors and is required for gastrulation and primitive streak formation, anchoring its developmental role.\",\n      \"evidence\": \"Gene targeting/knockout and chimera analysis in mice\",\n      \"pmids\": [\"9512518\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not distinguish which ligand(s) act through ALK4 at gastrulation\", \"Molecular basis of streak requirement not addressed\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Mapped signaling specificity to a seven-residue kinase subdomain IV\\u2013V loop, showing receptor specificity is encoded intracellularly rather than solely by ligand binding.\",\n      \"evidence\": \"Chimeric receptor construction and Xenopus animal cap assay\",\n      \"pmids\": [\"10075688\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of how the loop dictates SMAD/effector selection unknown\", \"Single lab, single study\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defined a disease-relevant dominant-negative mechanism whereby truncated ALK4 isoforms in pituitary tumors sequester type II receptors and block antiproliferative activin signaling.\",\n      \"evidence\": \"Reciprocal Co-IP, reporter and proliferation assays in transfected cells\",\n      \"pmids\": [\"11117535\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo contribution of truncated isoforms to tumorigenesis not tested\", \"Generality across tumor types unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showed ALK4 mediates Nodal signaling only with the co-receptor Cripto, and is the obligate type I receptor for a broad set of mesoderm-inducing ligands, defining its ligand range.\",\n      \"evidence\": \"Receptor reconstitution, Co-IP, and dominant-negative epistasis in Xenopus; somatic mutation sequencing in pancreatic cancer\",\n      \"pmids\": [\"11485994\", \"11248065\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the Nodal\\u2013Cripto\\u2013ALK4 complex not resolved at this stage\", \"Functional consequence of pancreatic somatic mutations on receptor activity not directly tested\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Established Cripto-1 as a direct ALK4-binding co-receptor that gates Nodal-dependent SMAD2 phosphorylation, while Cripto's MAPK/AKT activation is ALK4-independent.\",\n      \"evidence\": \"Phage display, Co-IP, FACS, and reporter assays in mammalian epithelial cells\",\n      \"pmids\": [\"11909953\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Domain-level interface not yet defined\", \"Quantitative affinity not measured\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Provided a selective pharmacological tool by showing SB-431542 inhibits ALK4/5/7 but not other ALKs or MAPK pathways, enabling specific dissection of SMAD2/3 signaling.\",\n      \"evidence\": \"In vitro kinase assays and cell-based signaling readouts\",\n      \"pmids\": [\"12065756\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cannot distinguish ALK4 from ALK5/ALK7 within the pathway\", \"No structural basis for selectivity provided\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the activin-A binding surface on ALK4 at the residue level and confirmed binding requires the type II receptor, linking structure to ligand engagement.\",\n      \"evidence\": \"Site-directed mutagenesis, dominant-negative assays, and 125I-activin-A crosslinking\",\n      \"pmids\": [\"12665502\", \"12649175\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No co-crystal structure of the ternary ligand\\u2013receptor complex\", \"Whether the same surface engages other ligands not tested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Confirmed ALK4 as the type I receptor selectively engaged by activin-A and myostatin (but not TGF-\\u03b2) and demonstrated ligand-specific use of ALK4 in left-right axis determination.\",\n      \"evidence\": \"Activin-A mutant crosslinking and antagonism assays; Co-IP and dominant-negative/morpholino epistasis in Xenopus\",\n      \"pmids\": [\"15123686\", \"15063168\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism that channels Vg1 but not Xnr1/derriere to ALK4 unknown\", \"Type II receptor partner specificity not dissected\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Characterized the Cripto CFC\\u2013ALK4 interface structurally, identifying the micromolar binding mode and key exposed residues.\",\n      \"evidence\": \"Synthetic CFC domain, NMR, SPR binding, and molecular docking\",\n      \"pmids\": [\"19035567\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No cellular mutagenesis validation of docking-predicted contacts\", \"Affinity is low/micromolar, physiological relevance of isolated domain unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated a tissue-homeostatic role: keratinocyte ALK4 is required for hair follicle cycling and restrains epidermal proliferation.\",\n      \"evidence\": \"Keratinocyte-specific conditional knockout with histology\",\n      \"pmids\": [\"21191412\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ligand and SMAD effector driving the phenotype not identified\", \"Molecular target genes not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved a complete signaling-to-phenotype axis for trophoblast invasion, with ALK4\\u2013SMAD2/3\\u2013SMAD4 upregulating SNAIL and then MMP2.\",\n      \"evidence\": \"siRNA knockdown of multiple pathway components, expression analysis, and Matrigel invasion assays\",\n      \"pmids\": [\"26305619\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance to placentation not tested\", \"Direct SMAD binding to SNAIL promoter not shown here\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established ALK4 as a driver of pathological fibrosis, with ALK4-deficient mice protected from atrial fibrosis and arrhythmia.\",\n      \"evidence\": \"ALK4 haplodeficiency mouse model with electrophysiology and histology\",\n      \"pmids\": [\"28639003\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-autonomous versus paracrine contribution not fully separated\", \"Downstream profibrotic transcriptional targets not defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed dual, context-dependent ALK4 roles in muscle, where knockdown enhances myoblast differentiation in vitro yet promotes atrophy in vivo.\",\n      \"evidence\": \"Antisense oligonucleotide knockdown with in vitro/in vivo functional readouts and RNAseq in mdx mice\",\n      \"pmids\": [\"27733450\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation of opposing in vitro/in vivo effects mechanistically unresolved\", \"Contribution of redundant type I receptors not addressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined neuronal ALK4 mechanisms in the nucleus accumbens, showing SMAD2-driven SATB1 nuclear repositioning controls somatostatin interneuron specification.\",\n      \"evidence\": \"Conditional knockout, Co-IP, nuclear fractionation, and immunostaining\",\n      \"pmids\": [\"31676717\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream ligand in MSNs not defined\", \"Direct SMAD2\\u2013SATB1 binding interface not mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated SMAD2/3 effector specificity and SMAD-independent ALK4 output, with distinct SMAD2-, SMAD3-, and SMAD-independent programs across cell types.\",\n      \"evidence\": \"siRNA knockdown, ChIP, nuclear fractionation, and functional assays in trophoblast, neuroblastoma, granulosa, and renal cells\",\n      \"pmids\": [\"33230889\", \"20226172\", \"31185247\", \"30804470\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis selecting SMAD2 vs SMAD3 vs SMAD-independent output unknown\", \"Many contexts rely on a single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established ALK4 as a redundant brake on skeletal muscle growth: combined Tgfbr1/Acvr1b deletion triggers hypertrophy via Akt/p70S6K and reduced E3 ligases, while single deletion does not.\",\n      \"evidence\": \"Muscle-specific double conditional knockout with phospho-Western, histology, and injury models\",\n      \"pmids\": [\"35323108\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligand redundancy upstream not fully resolved\", \"Whether SMAD or non-SMAD signaling enforces the brake not separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Expanded ALK4's homeostatic roles to adipose hyperplasia, with ALK4 suppressing precursor differentiation through CEBP\\u03b1/PPAR\\u03b3, and consolidated cocaine-related \\u0394FosB induction via SMAD3\\u2013PCBP1.\",\n      \"evidence\": \"Conditional knockout with rescue (CEBP\\u03b1 knockdown); conditional KO, Co-IP, RIP, and behavioral sensitization\",\n      \"pmids\": [\"36403856\", \"35730718\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligand driving adipose precursor ALK4 signaling not identified\", \"How D1 signaling converges on ALK4/SMAD3 mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established ALK4 as a Kras-cooperating pancreatic tumor suppressor whose loss promotes precancerous lesions from acinar and ductal cells, accelerated by pancreatitis.\",\n      \"evidence\": \"Cell-type-specific conditional knockouts with MRI and histopathology\",\n      \"pmids\": [\"39111635\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular tumor-suppressive mechanism not defined in this study\", \"Progression to invasive carcinoma not quantified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided a molecular mechanism for ALK4 tumor suppression: ALK4 loss raises MGAT5-dependent N-glycosylation and galectin-3-mediated stabilization of surface TGF-\\u03b2 receptors, amplifying canonical TGF-\\u03b2 signaling and progression.\",\n      \"evidence\": \"In vitro KO/knockdown, in vivo cancer models, glycosylation analysis, and MGAT5/galectin-3 rescue experiments\",\n      \"pmids\": [\"41408046\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How ALK4 controls MGAT5 expression not mechanistically resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Refined the Nodal signaling architecture, showing Cripto-1 bridges Nodal\\u2013ActRIIB to ALK4 via distinct EGF-like and CFC domains, contrasting with canonical ligand-bridging.\",\n      \"evidence\": \"AlphaFold3 modeling, SPR, domain-specific antibodies, and SMAD2/3 assays in NTERA-2 cells\",\n      \"pmids\": [\"39840816\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mutagenesis validation of modeled interfaces\", \"No experimental structure of the ternary complex\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved what determines ALK4's choice among SMAD2, SMAD3, and SMAD-independent outputs across tissues, and how upstream ligand and type II receptor combinations select these programs.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying structural/biochemical model of effector selection\", \"Tissue-specific ligand inputs largely inferred rather than mapped\", \"Cause-effect link between glycosylation control and SMAD output not integrated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 16]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [12, 16]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [4, 6, 8]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 6, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 16]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [5, 31, 33]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [16, 20, 26]}\n    ],\n    \"complexes\": [\n      \"ALK4\\u2013type II activin receptor (ActRII/IIB) heteromeric receptor complex\",\n      \"Nodal\\u2013Cripto-1\\u2013ALK4\\u2013ActRIIB ternary signaling complex\"\n    ],\n    \"partners\": [\n      \"ACVR2B\",\n      \"TDGF1\",\n      \"SMAD2\",\n      \"SMAD3\",\n      \"SMAD4\",\n      \"TGFBR1\",\n      \"SATB1\",\n      \"PCBP1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}