Drosophila Epsin's role in Notch ligand cells requires three Epsin protein functions: The lipid binding function of the ENTH domain, a single Ubiquitin interaction motif, and a subset of the C-terminal protein binding modules

Abstract

Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. Epsin’s mechanism of function is complex and context-dependent. Consequently, how Epsin promotes ligand endocytosis and thus Notch signaling is unclear, as is why Notch signaling is uniquely dependent on Epsin. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, we tested each of the five protein or lipid interaction modules for a role in Notch activation by each of the two ligands, Serrate and Delta. There are five main results of this work that impact present thinking about the role of Epsin in ligand cells. First, we discovered that deletion or mutation of both UIMs destroyed Epsin’s function in Notch signaling and had a greater negative impact on Epsin activity than removal of any other module type. Second, only one of Epsin’s two UIMs was essential. Third, the lipid-binding function of the ENTH domain was required only for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins were necessary collectively for Epsin activity, their functions were highly redundant; most unexpected was the finding that Epsin’s Clathrin binding motifs were dispensable. Finally, we found that signaling from either ligand, Serrate or Delta, required the same Epsin modules. All of these observations are consistent with a model where Epsin’s essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. We propose that Epsin’s specificity for Notch signaling simply reflects its unique ability to interact with the plasma membrane, Ubiquitin, and proteins that bind Clathrin.

Notch Ligand Endocytosis Generates Mechanical Pulling Force Dependent on Dynamin, Epsins, and Actin

“…Nonetheless, mouse embryos lacking both epsin1 and epsin2 display classic Notch mutant phenotypes, likely reflecting a role for epsins in ligand signaling activity and underscoring the absolute requirement for epsins in Notch-dependent events. Furthermore, since Dll1 ubiquitylation and epsin UIMs are required for Dll1-epsin complex formation, Dll1 cell-mediated force and Delta signaling activity in flies (Xie et al, 2012), we hypothesize recruitment of epsins by ubiquitylated ligands is critical for endocytic force to activate Notch.…”

Endocytosis and control of Notch signaling

“…Epsin also has motifs for interaction with AP2 and clathrin. In a recent study, one of the two UIMs, and the ENTH domain have been shown to be essential for Notch signaling [54]. In the same study, the clathrin binding motifs on the C-terminal region were dispensable although the extended region encompassing these and motifs for interaction with AP2 were necessary for Epsin function in Notch signaling.…”

Ubiquitinations in the Notch Signaling Pathway

“…The factor accounting for ubiquitinated ligand recognition is Epsin (Lqf in Drosophila ), a clathrin-interacting endocytic adaptor that is thought to bind the ligands through its ubiquitin interacting motif (UIM) [108,112–114]. Epsin’s role in ligand-emitting cell is rather complex and requires three epsin functional domains [114]: the ENTH (Epsin- N -Terminal Homology) domain binds PIP2 (phosphatidylinositol 4,5-biphosphate), inserts into the plasma membrane and induces membrane curvature. Then the epsin C -terminus recruits clathrin directly and also AP-2 and/or Eps15 to eventually promote clathrin-dependent ligand endocytosis.…”