Cripto-1 acts as a molecular bridge linking nodal to ALK4 via distinct structural domains.

The TGF-β family ligand Nodal is an essential regulator of embryonic development, orchestrating key processes such as germ layer specification and body axis formation through activation of SMAD2/3-mediated signaling. Significantly, this activation requires the co-receptor Cripto-1. However, despite their essential roles in embryogenesis, the molecular mechanism through which Cripto-1 enables Nodal to activate the SMAD2/3 pathway has remained elusive. Intriguingly, Cripto-1 also has been shown to antagonize other TGF-β family ligands, raising questions about its diverse functions. To clarify how Cripto-1 modulates TGF-β signaling, we integrated AlphaFold3 modeling, surface plasmon resonance (SPR)-based protein-protein interaction analysis, domain-specific anti-Cripto-1 antibodies, and functional studies in NTERA-2 cells. In contrast to canonical TGF-β signaling, where ligands bridge type I and type II receptors for activation, Nodal, bound to the type II receptor, utilizes Cripto-1 to recruit the type I receptor ALK4, forming a unique ternary complex for SMAD2/3 activation. Our molecular characterization of Cripto-1-mediated Nodal signaling clarifies the unique role of this enigmatic co-receptor and advances our understanding of signaling regulation within the TGF-β family. These insights have potential implications for both developmental biology and cancer research.