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

Abstract

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. On the other hand, a chimeric molecule encompassing the extracellular domain of Dll1 and the transmembrane/intracellular domain of Dll3, which contains no lysine, is endocytosed, recycled, and interacts with Notch1 but is unable to induce transendocytosis of the extracellular region of Notch1 or to signal. These observations suggest that the chimera uses an ubiquitination-independent signal to recycle, allowing it to acquire affinity for Notch1. Our results support the idea that ligand recycling determines its competence to bind efficiently to the receptor but that this is insufficient to allow it to perform transendocytosis, an event required for activation of Notch signaling. Finally, the present study indicates that Dll1 partially localizes to lipid microdomains, whereas both ubiquitination-defective Dll1 and the Dll1-3 chimera are excluded from these compartments, suggesting that these microdomains provide the environment necessary for Dll1 to activate Notch signaling.ubiquitination ͉ recycling ͉ transendocytosis ͉ membrane microdomains

Role of Recycling, Mindbomb1 Association, and Exclusion from Lipid Rafts of Delta-like 4 for Effective Notch Signaling To Drive T Cell Development

“…However, it remains unclear how both Dll1 and Dll4 are able to quantitatively induce different Notch signals, but are still able to support T cell development in vitro, when expressed at high levels.not induce receptor trans-endocytosis. In keeping with this, OP9 cells expressing this Dll1 mutant were unable to support T cell development in vitro (23), suggesting that Dll1 may require endocytosis as a mechanism for efficient T cell development. Currently there are no data on the requirement of Dll4 endocytosis during T cell development.…”

“…Endocytosis of the Notch ligands has been proposed to activate the Notch receptor (41). Recent work has explored the endocytosis of Dll1 (23,40), but to date the role of Dll4 endocytosis for its function is unknown. In this study, we examined the role of endocytosis of Dll4 and its ability to support T cell development.…”

“…In this study, we examined the role of endocytosis of Dll4 and its ability to support T cell development. The recycling model posits that endocytosis of Notch ligands allows their ECD to undergo posttranslational modifications and recycle back to the cell surface with an enhanced ability to interact with Notch receptors (6,23). Internalization of Notch ligands is mediated by ubiquitination of their intracellular domain by E3 ubiquitin ligases: Mib and Neur (7)(8)(9)(10).…”

“…Recently, Heuss et al (23) have shown that Dll1 is localized to lipid rafts. We used sucrose-density gradient to assess the localization of Dll4 and DL4-CD25 constructs.…”

Endocytosis in Notch Signaling Activation

“…Rescue of epsin absence was achieved by expressing a chimeric DSL, in which the intracellular tail was replaced by a short internalization signal of the LDL receptor, which was known to mediate the internalization and recycling of many proteins through the endosome [172]. Further studies in Drosophila and in mammalian cells have substantiated this initial observation of the existence of a possible trafficking step for the maturation of Notch ligands: (i) a defect in Delta trafficking through the recycling endosome was proposed to cause the aberrant cell fate transformation in sec15 mutant sensory lineages (see next paragraph for links of this developmental pathway to Notch signaling) [173]; (ii) expression of a dominant negative Rab11 (a small GTPase which regulates trafficking from the recycling endosome to the PM) was associated with DSL accumulation in endosomes and Notch signaling failure in a mammalian co-culture system [174]; (iii) an ubiquitylation-defective mutant of Dll1 can be efficiently endocytosed, but in contrast to the wild-type isoform is unable to recycle back to the cell surface and, possibly as a consequence of this trafficking defect, to efficiently bind Notch1 in a mammalian cell system [175].…”