DLK1 Blocks Muscle Aging Receptor ACVR2B, Not Notch as Long Believed

DLK1 (Delta-like non-canonical Notch ligand 1) has long been assumed to regulate development—including myogenesis, adipogenesis, and neurogenesis—primarily by inhibiting Notch signaling. However, the molecular basis for this assumption rested largely on indirect evidence and yeast two-hybrid data generated under non-physiological conditions. This study challenges that paradigm at the structural and functional level.

Using surface plasmon resonance (SPR), flow cytometry, and fluorescent reporter assays, the authors definitively showed that DLK1 does not bind NOTCH1, NOTCH2, or NOTCH3, and that neither immobilized nor soluble DLK1 activates or inhibits ligand-mediated Notch signaling. Sequence analysis confirmed that DLK1 lacks the C2 and DSL domains required for canonical Notch ligand interactions, and key hydrophobic Notch-binding residues in JAG1 are replaced by smaller amino acids in DLK1.

Mining the Bioplex 3.0 protein-protein interaction database, the researchers identified ACVR2B—a TGF-β superfamily type II receptor—as the sole known cell surface receptor among DLK1's candidate binding partners. SPR confirmed a direct interaction with a dissociation constant (KD) of ~1.4 µM. A yeast surface-display screen against 12 TGF-β superfamily receptors showed DLK1 binds exclusively to ACVR2B, demonstrating remarkable selectivity compared to promiscuous TGF-β ligands like Activin or BMP-2.

The crystal structure of the DLK1 EGF4-6 fragment in complex with the ACVR2B extracellular domain revealed that DLK1 engages the canonical ligand-binding site of ACVR2B. EGF5 of DLK1 inserts a hydrophobic 'finger loop' into a conserved hydrophobic cleft on ACVR2B—the same site used by myostatin and Activin. In functional assays, DLK1 antagonized myostatin-ACVR2B signaling (blocking SMAD2/3 phosphorylation) and promoted myoblast differentiation in C2C12 cells, phenocopying established effects of ACVR2B inhibition in muscle.

Finally, the authors showed that DLK1's indirect influence on Notch outputs is mediated through disruption of SMAD2/3 and Notch intracellular domain (NICD) co-localization—a known crosstalk mechanism—rather than through direct Notch receptor binding. This reconciles conflicting reports linking DLK1 to Notch phenotypes without requiring a direct physical interaction. Therapeutically, these findings position DLK1 as a natural endogenous brake on myostatin/Activin signaling with implications for muscle wasting, sarcopenia, and cancer cachexia.