Ligand-activated Notch undergoes DTX4-mediated ubiquitylation and bilateral endocytosis before ADAM10 processing.

The Notch signaling pathway, which is activated by cell-cell contact, is a major regulator of cell fate decisions. Mammalian Notch1 is present at the cell surface as a heterodimer of the Notch extracellular domain associated with the transmembrane and intracellular domains. After ligand binding, Notch undergoes proteolysis, releasing the Notch intracellular domain (NICD) that regulates gene expression. We monitored the early steps of activation with biochemical analysis, immunofluorescence analysis, and live-cell imaging of Notch1-expressing cells. We found that, upon ligand binding, Notch1 at the cell surface was ubiquitylated by the E3 ubiquitin ligase DTX4. This ubiquitylation event led to the internalization of the Notch1 extracellular domain by the ligand-expressing cell and the internalization of the membrane-anchored fragment of Notch1 and DTX4 by the Notch1-expressing cell, which we referred to as bilateral endocytosis. ADAM10 generates a cleavage product of Notch that is necessary for the formation of the NICD, which has been thought to occur at the cell surface. However, we found that blocking dynamin-mediated endocytosis of Notch1 and DTX4 reduced the colocalization of Notch1 with ADAM10 and the formation of the ADAM10-generated cleavage product of Notch1, suggesting that ADAM10 functions in an intracellular compartment to process Notch. Thus, this study suggests that a specific pool of ADAM10 acts on Notch in an endocytic compartment, rather than at the cell surface.