This lack of overlap between dAux and Clc seems more consistent with the notion that auxilin is required for the dissociation of clathrin coats from CCVs under physiological conditions. Our analysis of clearly suggests that auxilin plays an important role in the Notch cascade in multiple Loxoprofen Notch-dependent processes. protein is disrupted in auxilin mutant tissues. Thus, our data suggest that auxilin Loxoprofen is an integral component of the Notch signaling pathway, participating in the ubiquitin-dependent endocytosis of Delta. Furthermore, the fact that auxilin is required for Notch signaling suggests that ligand endocytosis in the signal-sending cells needs to proceed past coat disassembly to activate Notch. Introduction Endocytosis, a process characterized by the internalization of extracellular materials and membrane proteins via vesicular intermediates, plays many roles in regulating cellCcell signaling pathways. In addition to the well-established role of attenuating signaling activity by clearing active receptor molecules from the cell surface, endocytosis has been proposed to facilitate signaling by transporting active receptor molecules to sites where downstream effectors are localized (Entchev et al., 2000; Dubois et al., 2001; Sorkin and Von Zastrow, 2002). A novel role of endocytosis has recently been proposed for the Notch signaling cascade, in which the internalization of the ligand facilitates activation of the receptor (Lai, 2004; Le Borgne et al., 2005a), although the exact mechanism of this critical event remains elusive. The Notch pathway is a signaling module that is highly conserved in all metazoans and has been implicated in a variety of developmental processes (Artavanis-Tsakonas et al., 1999). How Notch transduces signals from the plasma membrane and IL10RB affects gene regulation has been extensively analyzed in Loxoprofen dynamin, a GTPase required for the detachment of vesicles from plasma membrane (Kosaka and Ikeda, 1983; van der Bliek and Meyerowitz, 1991), was found to produce a Notch-like phenotype (Poodry, 1990). Clonal analysis suggested that in Notch signaling, dynamin function is required in both signal-sending and signal-receiving cells (Seugnet et al., 1997), suggesting that endocytosis impinges on the pathway at two independent steps. Although the role of endocytosis in signal-receiving cells is less clear, the internalization of ligand for the Notch receptor in the signal-sending cells appears to be a key event in activating the Notch cascade (Parks et al., 2000). In Lag-2 protein family (DSL). Both Dl and Ser appear to use an ubiquitin-mediated endocytic pathway to activate Notch receptors (Lai et al., 2005; Loxoprofen Le Borgne et al., 2005b; Pitsouli and Delidakis, 2005; Wang and Struhl, 2005). The covalent addition of ubiquitin to polypeptides, besides being a tag for proteasome-mediated protein degradation, can serve as a sorting signal for membrane protein internalization (Hicke and Riezman, 1996; Terrell et al., 1998). The ubiquitination of Dl and Ser for subsequent internalization is mediated by (((homologue of epsin (Cadavid et al., 2000). contains an ubiquitin-interacting motif (Polo et al., 2002; Shih et al., 2002), as well as motifs that bind to clathrin and other classes of adaptors (Bonifacino and Traub, 2003). Thus, it is thought that lqf functions as a cargo-specific clathrin adaptor, capable of recognizing and sequestering monoubiquitinated DSL ligand into clathrin-coated vesicles (CCVs; Overstreet et al., 2003, 2004; Wang and Struhl, 2004), although an alternative function for epsin in nonclathrin endocytosis has been proposed (Chen and De Camilli, 2005; Sigismund et al., 2005). Although a requirement of ligand endocytosis for Notch activation seems clear, the mechanism of how the internalization of the DSL ligand in the signal-sending cells promotes the proteolytic processing of Notch in the neighboring signal-receiving cells remains poorly understood. One set of models proposed that the internalization of Notch bound DSL ligand could either clear NECD from the extracellular space or generate physical force to dissociate NECD from the membrane-tethered intracellular domain, allowing the subsequent cleavage processing to occur (Parks et al., 2000). Alternatively, it has been suggested that endocytosis is required to transport DSL Loxoprofen ligand to subcellular compartments, where the ligand is rendered signaling competent before being recycled back to.