Functional Analysis Of The Nhr2 Domain Of Neuralized Indicates That Oligomerization Regulates Ubiquitination And Endocytosis Of Notch Ligands.

The Notch pathway plays an integral role in development by regulating cell fate in a wide variety of multicellular organisms. A critical step in the activation of Notch signaling is the endocytosis of the Notch ligands, Delta and Serrate. Ligand endocytosis is regulated by one of two E3 ubiquitin ligases, Neuralized or Mind bomb. Neuralized (Neur) is comprised of a C-terminal RING domain, which is required for Delta ubiquitination, and two Neur homology repeat (NHR) domains. We have previously shown the NHR1 domain is required for Delta trafficking. Here we show that the NHR1 domain also affects binding and internalization of Serrate. Furthermore, we show that the NHR2 domain is required for Neur function and that a point mutation in the NHR2 domain (Gly430) abolishes Neur ubiquitination activity and affects ligand internalization. Finally, we provide evidence that Neur can form oligomers in both cultured cells and fly tissues, which regulate Neur activity and, by extension, ligand internalization.