A Glycosphingolipid Binding Domain Controls Trafficking and Activity of the Mammalian Notch Ligand Delta-like 1

The activity of Notch ligands is tightly regulated by trafficking events occurring both before and after ligand-receptor interaction. In particular endocytosis and recycling have been shown to be required for full signaling activity of the ligands before they encounter the Notch receptor. However little is known about the precise endocytic processes that contribute to ligand internalization. Here we demonstrate that endocytosis contributes to Dll1 signaling activity by preserving the ligand from shedding and degradation. We further show that the glycosphingolipid-binding motif originally identified in Drosophila Notch ligands is conserved in mammals and is necessary for Dll1 internalization. Mutation of its conserved tryptophan residue results in a Dll1 molecule which is rapidly inactivated by shedding and degradation, does not recycle to the cell surface and does not activate Notch signaling. Finally, silencing in the signal-sending cells of glucosylceramide synthase, the enzyme implicated in the initial phase of glycosphingolipid synthesis, down-regulates Notch activation. Our data indicate that glycosphingolipids, by interacting with Dll1, may act as functional co-factors to promote its biological activity.