The Notch pathway is highly conserved across invertebrates and vertebrates and plays multiple and essential roles in many developmental processes, such as inhibiting differentiation by lateral signaling and regulating cell fate through inductive interactions (7
). Notch signaling is induced through direct cell-cell interactions between membrane-bound Notch ligands, Delta, Serrate, and Lag-2 (DSL), and the Notch receptor on adjacent cells. The activation of the Notch receptor results in the cleavage of Notch by the γ-secretase complex, leading to the translocation of the Notch intracellular domain into the nucleus, where it can activate downstream target genes (7
). Recessive loss-of-function mutations in key components of the Notch pathway in Drosophila melanogaster
are embryonic lethal and result in neurogenic phenotypes consisting of an overgrowth of the nervous system at the expense of the epidermis (36
The ubiquitination and endocytosis of receptors and ligands have been shown to potentiate Notch signaling (32
). Currently, there are two models hypothesizing how ligand endocytosis facilitates Notch activation in the signal-receiving cell. The mechanical force or pulling model suggests that Delta endocytosis exerts a force on the Delta-Notch complex that alters the conformation and promotes the cleavage of the Notch extracellular domain (NECD), which is a critical step in Notch activation (17
). The recycling model suggests that the modification of an inactive form of Delta in an endosomal compartment makes Delta a more effective ligand, which will be re-presented to the cell surface (perhaps at a microdomain of the plasma membrane) to activate Notch (2
). In the signal-sending cell, Neuralized (Neur) (12
) and Mindbomb1 (3
) are two E3 ubiquitin ligases that regulate the endocytosis of the Notch ligands Delta and Serrate by ubiquitination.
Neur was one of the first five Notch pathway members identified (36
). Previous analyses revealed that Neur plays an important role in all three germ layers during embryonic development (10
). In addition, Neur is also required for the development of the adult central and peripheral nervous system, including bristle sense organ patterning and photoreceptor specification (29
). Consistent with its role in embryogenesis and adult neurogenesis, Neur is expressed in embryonic neural tissue and in the region of larval imaginal discs that will give rise to adult sensory organs (5
). Of note, Neuralized is not required for all Notch signaling events, and evidence suggests that Mind bomb, its functional homologue, performs the same role in different cellular and developmental contexts (28
). The presence of either Neur or Mind bomb in the signal-sending cell appears to be required for ligand endocytosis (34
). In addition to its role in Notch signaling, Neur was also recently shown to regulate epithelial cell polarity in the embryo (8
locus produces two major transcripts, neur-RA and neur-RC, which give rise to two proteins, NeurPA and NeurPC, which differ only at their N termini (9
). Specifically, NeurPA, which is the predominant isoform during development (5
), contains a phosphoinositide (PIP)-binding motif at the N terminus, which is required for Delta endocytosis downstream of Delta ubiquitination by Neur (49
). In addition, both isoforms contain three highly conserved domains, including a carboxyl-terminal RING domain and two Neuralized homology repeat (NHR) domains (NHR1 and NHR2). The RING domain is both necessary and sufficient for Neur E3 ubiquitin ligase activity and is required for the endocytosis of the Notch ligand Delta (27
). The NHR1 domain is a protein-protein interaction module that is required for Neur to bind Delta: a point mutation in a highly conserved glycine residue at position 167 disrupts Delta binding (9
). Whether the NHR1 domain also mediates the interaction between Neur and Serrate (44
) is unknown.
Although several aspects of the role of Neur in Notch signaling have been characterized, the full spectrum of Neur function and regulation still remains to be elucidated, including the function of the NHR2 domain. Previous in vitro
studies done with mammalian cell cultures demonstrated that the NHR2 domain of Neurl1, a mouse homologue of Drosophila
Neur, mediates the interaction between Neurl1 and Jagged1 (26
), a mouse homologue of Drosophila
Serrate. Whether the NHR2 domain is required for Neur function in vivo
and whether this function depends solely on its interaction with Serrate remain to be determined.
Here we show that Neur binds both Notch ligands via the NHR1 domain. We also show that the NHR2 domain is required for Neur-mediated Dl endocytosis and Notch signaling in vivo. Specifically, we show that the deletion of the NHR2 domain and a point mutation in a highly conserved glycine residue within the NHR2 domain reduce Dl internalization. Finally, we demonstrate that Neur can form oligomers, which regulate its ubiquitination activity and, as a result, ligand internalization.