-arrestin moves up a notchIntroduction
Non-visual forms of arrestin in mammals — 
-arrestin-1 and -arrestin-2, also known as arrestin-2 and arrestin-3, respectively — were originally described as molecules that desensitize seven-transmembrane-spanning receptors in conjunction with G-protein-coupled receptor kinases. However, it is now apparent that they also serve as multipurpose endocytic and signalling adaptors and that they act on other types of receptor such as the tyrosine kinase, insulin-like growth factor 1 receptor (IGF1R)1. On page 1191 of this issue, Mukherjee et al. demonstrate that Kurtz, the unique, non-visual arrestin of Drosophila, regulates ubiquitin-dependent downregulation of the single transmembane-spanning receptor, Notch. Kurtz binds Deltex (a known Notch regulator and a putative E3 ubiquitin ligase for Notch), thus promoting ubiquitination and degradation of Notch2.
In multicellular organisms, cell-fate decisions are crucial for the progressive differentiation of precursor cells during normal organogenesis3. Notch signalling has a central role in this process: communication between adjacent cells occurs through a 'juxtacrine' signalling pathway in which one cell expresses the cell surface receptor (Notch) and a neighbouring cell expresses a Notch ligand (such as the transmembrane protein, Delta or Serrate–Jagged) on its surface. Ligand activation causes the Notch protein to be proteolysed, leading to the release of its intracellular domain, which then enters the nucleus where it functions as a transcriptional cofactor that activates downstream gene transcription (Fig. 1a). The outcome of Notch-mediated signalling is determined by the amounts of receptor and ligand in apposing cells, both of which are precisely regulated. As the basis of Notch-dependent cell–cell communication depends on the differential expression of receptor and ligand in juxtaposed cells, even small changes in the levels of either protein are amplified as a developmental signal. This fine regulation is further controlled by the fact that cells that are exposed to high levels of either Notch or Delta produce very little of the complementary protein. Typically, Delta-expressing cells lack the transcriptional activity of Notch, forcing them to enter a specific developmental pathway such as a neuronal fate. On the other hand, cells expressing Notch follow a path to become ectodermal cells and are excluded from a neuronal fate, presumably due to Notch-dependent transcription (Fig. 1a). The Notch pathway is evolutionarily conserved in many metazoans, including worms and mammals.
Figure 1: Notch signalling and degradation (a) Extracellular domains of the Notch receptor and its ligand, Delta, interact and lead to Notch activation, as well as proteolytic cleavage of Notch at the cell membrane.
This releases the intracellular domain of Notch which translocates into the nucleus, where it activates gene transcription. The cell is then driven to an ectodermal fate. In the apposing cell, Delta and bound extracellular domains of Notch are internalized into vesicles and are subsequently degraded. These events somehow cause this cell to develop into a neuronal cell. (b) Deltex, a ubiquitin ligase, binds to the non-visual arrestin, Kurtz. This complex interacts with the intracellular domains of Notch. Deltex efficiently ubiquitinates Notch only when complexed with Kurtz. Degradation of ubiquitinated Notch seems to be dependent on the 26S proteasome in Drosophila S2 cells. It is highly likely that this ternary complex may possess other functional capabilities, depending on the particular cell or tissue type. Other putative Kurtz binding partners are indicated by the green triangle.
Full size image (58 KB)Mukherjee et al. provide insights into Notch regulation. Notch is a large protein with a single transmembrane domain, many epidermal growth factor-like repeats in the extracellular domain, and motifs that regulate Notch function, ubiquitination and degradation in the intracellular domain. Substrate ubiquitination is carried out by specialized enzymes (called E3 ubiquitin ligases) either directly, or through adaptor proteins, and typically targets proteins for degradation by the 26S proteasome. This work describes Notch ubiquitination by Deltex, an E3 ubiquitin ligase. Remarkably, the authors identify Kurtz as a Deltex-binding protein and show that the presence of Kurtz, which is probably functioning as an E3 adaptor, facilitates the process of Notch ubiquitination and degradation. Loss of Kurtz alone increased Notch levels, whereas increasing both Kurtz and Deltex expression was required to diminish Notch protein levels and produce the characteristic phenotype of 'notching' around the Drosophila wing margin.
-arrestin-2 has analogous roles in the ubiquitination of the 2-adrenergic and V2 vasopressin seven-transmembrane-spanning receptors by as yet unidentified E3 ligases4, 5. Recently, -arrestin-1 was also shown to function as an adaptor that recruits the ubiquitin ligase Mdm2 to ubiquitinate the IGF1R6. For the seven-transmembrane-spanning receptors, degradation occurs in lysosomes, whereas proteasomes perform this function for the IGF1R. The authors showed that the Kurtz–Deltex–Notch ternary complex is internalized into membrane-vesicular compartments, although the precise identity of these vesicles remains to be determined. In fact, Mukherjee et al. show that chemical inhibition of proteasomal activity leads to Notch stabilization, suggesting proteasomal degradation of Notch. However, lysosomal-versus-proteasomal degradation of Notch may depend on the cellular context, as ubiquitination and lysosomal degradation of mammalian Notch by the c-Cbl ubiquitin ligase in a skeletal myoblast cell line has been reported7.
Although Notch and Deltex can interact directly, recruitment of Kurtz to this complex leads to marked enhancement of Notch ubiquitination2. Perhaps Kurtz recruits additional enzymatic machinery to facilitate this process. In addition to binding Deltex and Mdm2, -arrestins may interact with other E3 ligases. In fact, other ubiquitin ligases have been implicated in ubiquitinating Notch8, 9. Thus, Notch ubiquitination and downregulation may be regulated by redundant mechanisms involving multiple families of E3 ligases, and -arrestin may also regulate these processes.
Ubiquitination of mammalian seven-transmembrane-spanning receptors facilitated by -arrestin is an agonist-stimulated modification that leads to receptor degradation in lysosomal compartments. In contrast, the effects of Kurtz and Deltex on Notch trafficking and ubiquitination2 are unaffected by the levels of its ligand, Delta. It is plausible that other factors (for example, other ligands) may regulate the formation of the Notch–Deltex–Kurtz ternary complex, and that the interaction of Deltex and Kurtz with Notch might have additional consequences. Deltex-dependent, but ligand-independent, Notch signalling has also been reported during Drosophila myogenesis and neurogenesis10. This Notch activity, mediated by a cytoplasmic pathway, restricts cells from acquiring neural or myogenic competence, and requires the activity of Deltex and the serine-threonine kinase GSK3. What role, if any, Kurtz may have in Deltex-dependent Notch signalling awaits further biochemical characterization.
The binding affinity of mammalian -arrestins for various receptors is largely dictated by their phosphorylation1. The intracellular domain of Notch contains many phosphorylation motifs, raising the possibility of a direct interaction between -arrestin and phosphorylated Notch. Another important property of the interaction between -arrestin and seven-transmembrane-spanning receptors is that it triggers ubiquitination of the -arrestin proteins themselves, which then facilitates receptor internalization and stabilizes compartmentalization of activated ERK-1 and -2, associated with the receptor--arrestin signalling scaffolds11. Whether Kurtz performs similar roles in Notch signalling or endocytosis and whether it can itself be ubiquitinated in the Notch–Deltex–Kurtz ternary complex remain to be determined.
-arrestins have important roles as adaptors during clathrin-mediated endocytosis of seven-transmembrane-spanning receptors1. Previous studies have shown Deltex-dependent trafficking and stabilization of Drosophila Notch in late endosomes12. The details of Notch signalling from these membrane compartments have yet to be clarified. Paradoxically, in the Notch system endocytosis seems more influential in the ligand-expressing cell (signal-sending cell) than in the receptor (Notch)-expressing cell (signal-receiving cell)13, 14. Activation of the released intracellular domain of Notch requires that the extracellular segment of Notch that is bound to the ligand (such as Delta) is internalized by the neighbouring cell. Inhibition of this 'trans-endocytosis' prevents activation of the Notch intracellular domain in the apposing cell. Furthermore, endocytosis-defective Delta proteins cannot induce Notch signalling. Ubiquitination also modifies Notch ligands and influences their endocytic sorting. It is tantalizing to imagine how Kurtz contributes to these processes as an endocytic and/or ubiquitination adaptor.
Signalling mechanisms mediated by other receptors and ligands (such as Wnt/wingless, TGF-/BMP, sonic Hedgehog, receptor tyrosine kinases and nuclear receptors) also participate in developmental processes, and mammalian -arrestins are known to regulate several of these systems. For example, mammalian -arrestin-2 mediates activity-dependent internalization of Smoothened receptors and complexes with Dishevelled to regulate endocytosis of a Wnt-5A–Fz4 receptor complex1. Interestingly, Notch activity, mediated by Deltex, represses neural fates and is antagonized by Dishevelled, a component of the Wingless signalling pathway15. Given the role of -arrestin in Notch signalling and its known interaction with Dishevelled, it is tempting to speculate that it may act in this crosstalk mechanism.
Notch signalling has a fundamental role in cell fate decisions in the nervous, vascular and haematopoietic systems. Aberrant Notch signalling is associated with tumorigenesis and has been shown to affect neurogenesis, angiogenesis and lymphoid development. To control the precise levels of signalling required for normal development and homeostasis, it is essential that cell membrane levels of Notch be tightly regulated. Deltex-dependent Notch ubiquitination facilitated by Kurtz provides a new mechanism for terminating the nuclear activity of Notch, and also regulates its cell-surface expression. Whether this mechanism exists simply to downregulate Notch activity or whether, similarly to mammalian -arrestin, it also serves to mediate aspects of Notch signalling (Fig. 1b), requires futher investigation.
