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.

The intracellular region of Notch ligands Dll1 and Dll3 regulates their trafficking and signaling activity.

Genetic studies have shown that ubiquitination and endocytosis of the Drosophila ligand Delta in signal-sending cells are required for activation of Notch signaling, but how these events promote Notch activation remains poorly understood. Here, we show that an ubiquitination-defective mutant of the murine Delta-homologue Dll1 is endocytosed but, in contrast to the wild-type Dll1, is unable to subsequently recycle back to the cell surface or to bind Notch1 efficiently. These results demonstrate that ubiquitination, although not required for endocytosis, is essential for Dll1 recycling and that recycling is required to acquire affinity for the receptor.

The notch ligand Delta1 recruits Dlg1 at cell-cell contacts and regulates cell migration.

Delta1 acts as a membrane-bound ligand that interacts with the Notch receptor and plays a critical role in cell fate specification. By using peptide affinity chromatography followed by mass spectrometry, we have identified Dlg1 as a partner of the Delta1 C-terminal region. Dlg1 is a human homolog of the Drosophila Discs large tumor suppressor, a member of the membrane-associated guanylate kinase family of molecular scaffolds.

Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor.

Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu.