Current Biology, Vol. 13, R273–R275, April 1, 2003, ©2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0960-9822(03)00199-4

Notch Signaling: Endocytosis Makes Delta Signal Better Roland Le Borgne and François Schweisguth

Endocytosis of cell surface receptors is involved in down-regulation of receptor activity. Recent findings indicate that, paradoxically, endocytosis of a membrane-spanning ligand may up-regulate receptor activity: the zebrafish E3 ligase Mind bomb promotes the endocytosis of Delta and is required for efficient activation of Notch.

The regulation of signaling by many cell surface receptors is intimately linked to membrane trafficking [1]. Internalization of active receptors is a well-known mechanism of densensitization that involves trafficking of active receptors to the lysosome, where they are degraded. Receptor endocytosis may also be required for signal transduction, possibly by bringing active receptors to a specific intracellular compartment where they associate with their signaling machinery. By contrast, little is known about the functional significance of the endocytosis of transmembrane ligand molecules. Recent studies [2–6] suggest that endocytosis of Delta family ligands is required for activation of Notch receptors in both flies and zebrafish. Signaling by Notch receptors involves three successive cleavages [7] (Figure 1). Notch is first processed in the trans-Golgi network, where cleavage at the extracellular ‘S1’ site produces a functional cell surface receptor. A second, ligand-dependent cleavage of Notch occurs at the extracellular ‘S2’ site and generates a membrane-bound activated form, called ‘NEXT’. The NEXT fragment is then processed at an intramembraneous ‘S3’ site to release the active ‘Notch intracellular domain (NICD), which acts as a transcriptional regulator. Notch signaling regulates numerous cell-fate decisions during development. In the neural plate of zebrafish, cell–cell interactions mediated by Notch are important to select early neural progenitor cells from neurogenin1 (ngn1)-expressing cells. In mind bomb (mib) mutant embryos, too many cells express high levels of ngn1 and prematurely become neurons. In a recent paper, Itoh et al. [3] have shown that this phenotype results from defective Notch signaling. The mib mutant phenotype can be rescued by injecting an activated form of Notch5, Notch5ICD, indicating that mib activity is required at the level or upstream of S3 cleavage. Also, expression of all four zebrafish homologues of Delta is up-regulated in mib mutant embryos, indicating that defective Notch signaling is not due to a loss in Delta expression. Positional cloning of the mib gene revealed that it encodes a novel, evolutionarily conserved E3 ubiquitin CNRS UMR 8542, Département de Biologie, Ecole Normale Supérieure, 46, rue d’Ulm 75230 Paris cedex France. E-mail: [email protected]

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ligase. Ubiquitin is a 76-amino acid polypeptide that is covalently linked to substrates in a multi-step process that involves a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a ubiquitin-protein ligase (E3). E3s recognize specific substrates and catalyze the transfer of ubiquitin to the protein substrate. Ubiquitin was first identified as a tag for proteins destined for degradation. More recently, ubiquitin has also been shown to serve as a signal for endocytosis [8]. One class of E3s, which includes Mib, is characterized by the presence of a catalytic RING domain. All five mib mutant alleles are loss-of-function mutations that affect the prototypical RING domain, demonstrating the importance of this domain for Mib function. Itoh et al. [3] have shown that the RING domain of Mib has E3 ligase activity in vitro and have provided strong evidence that Delta is the key target of Mib in zebrafish embryos. This is indicated by the finding that Xenopus Delta1 (XDelta1) physically interacts with Mib in transfected cells, and that it can be ubiquitinated by Mib in a reaction that is dependent on an intact RING domain. Moreover, internalization of XDelta1 into a late endosomal compartment requires both the intracellular domain of XDelta1 and the RING domain of Mib. Finally, in-frame fusion of a ubiquitin peptide to a dominant-negative form of XDelta1 that lacks its intracellular domain, XDelta1∆ICD, restored the protein’s ability to be endocytosed and to activate Notch in zebrafish embryos. Together, these results indicate that Mib regulates endocytosis of Delta during zebrafish development. They further indicate that endocytosis of Delta is required for Notch signaling. How does Mib-dependent endocytosis of Delta regulate Notch activation? Two models may be considered. One hypothesis is that Mib acts in signal-receiving

Delta NECD S2 Extracellular

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Figure 1. A model of Notch signaling. Delta at the surface of the signaling cell (top) binds S1 cleaved Notch at the surface of the responding cell (bottom). Liganddependent S2 cleavage of Notch generates NEXT, which is further processed at the S3 site.

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Figure 2. Possible models of the Miband Neur-dependent activation of Notch. Ubiquitination of Delta may promote: (A) the clustering of Delta-bound receptors; (B) the unmasking of the S2 site; (C) the clearance of NECD that would otherwise accumulate extracellularly; or (D) the formation within MVBs of signaling exosomes. See text for details.

A Ubiquitin

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C Ubiquitin

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cells to promote signal transduction, for instance by sorting Delta away from Notch at the surface of signalreceiving cells and thereby preventing cis-inhibition of Notch by Delta. This hypothesis was tested in C2C12 cells, in which increasing amounts of transfected XDelta1 inhibits activation of a Notch-responsive reporter construct. Transfection of Mib in these cells

S3

did not modulate the inhibitory effect of XDelta1. This negative result argues against this hypothesis. A second hypothesis is that Mib acts in signaling cells to up-regulate Delta activity. To test whether Mib acts in signal-receiving or in signal-emitting cells, cell transplantation was performed. Mosaic embryos composed of cells that are wild-type and of cells that

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are mib mutant, or that have reduced mib activity following morpholino-mediated inactivation, were analyzed. Detailed analysis of cell specification in the neural tube revealed that mib mutant cells are less likely to become neuron than their wild-type neighbours. This implies that mib mutant cells are able to receive the inhibitory Delta signal, but that they do not efficiently inhibit wild-type cells from adopting a neural fate. Thus, Mib-dependent endocytosis of Delta appears to be required to produce a strong inhibitory signal and to non-autonomously activate Notch in neighbouring cells. Strikingly, studies on the Drosophila and Xenopus gene neuralized (neur) have led to results that almost exactly parallel those described above for mib in zebrafish. Neur is an evolutionarily conserved RING finger-type E3 ubiquitin ligase which is required for Notch signaling in Drosophila. Neur interacts with Delta, promotes the ubiquitination of Delta and stimulates the accumulation of Delta into intracellular vesicles [2,4,6,9]. So, like Mib, Neur is thought to ubiquitinate an endocytic motif within the intracellular domain of Delta, thereby regulating Delta endocytosis. The mechanism by which Neur regulates Notch activity is unclear, however, in part because clonal analysis gave contradictory results [6,10,11]. Interestingly, the Neur and Mib proteins differ in domain composition, and both the neur and mib genes have been conserved during evolution. One hypothesis is that neur is the functional homologue of mib in flies and that these two genes have co-evolved with the intracellular domain of Delta, which is highly divergent between flies and vertebrates. Alternatively, neur and mib may have partially redundant functions. Consistently, injection of XDelta1 in mib mutant embryos suppressed neurogenesis [3], indicating that XDelta1 still signals in the absence of mib. It will thus be of interest to examine whether injection of zebrafish Neur can suppress the mib phenotype and, conversely, whether Neur is required for XDelta1 to signal in mib mutant embryos. A number of additional observations support the notion that Delta endocytosis facilitates Notch activation. First, genetic analysis in Drosophila indicates that dynamin-dependent endocytosis is required in signal-sending cells to promote Notch activation [12], possibly upstream of the S3 cleavage of Notch [13]. Second, endocytosis-defective Delta proteins have reduced signaling capacity [5]. Third, the membrane-bound extracellular domain of mouse Delta1 is not sufficient to activate Notch, suggesting that activity of the Delta1 intracellular domain is required upstream of the S3 cleavage [14]. How might Mib- or Neur-mediated endocytosis of Delta promote Notch activation? The primary effect of Mib (or Neur) may be to induce clustering of DeltaNotch complexes, thereby facilitating the extracellular S2 cleavage of Notch (Figure 2A). A second possibility is that endocytosis of Delta bound to Notch triggers a conformational change which facilitates S2 cleavage (Figure 2B) [5]. A third possibility is that endocytosis is required to clear the extracellular space of NECD, which would otherwise inhibit Notch activation by

titrating Delta (Figure 2C). These last two models are consistent with the observation that NECD is transendocytosed into signaling cells in Drosophila [5]. Lastly, ubiquitination of Delta might serve as a sorting signal to accumulate Delta at the surface of vesicles budding within the lumen of multi-vesicular bodies (MVBs). Upon release in the extracellular space, these hypothetical vesicles might act similarly as exosomes to activate Notch (Figure 2D). Further investigation on Mib and Neur will likely provide new and exciting insights into how membrane trafficking regulates developmental signaling.

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