Epsin is part of a protein complex that performs endocytosis in eukaryotes. Drosophila epsin, Liquid facets (Lqf), was identified because it is essential for patterning the eye and other imaginal disc derivatives. Epsins are endocytic proteins that bind phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] in the plasma membrane as well as Clathrin, AP-2 Adapter Complex, and other accessory proteins in coated pits (reviewed by Wendland, 2002). Epsins were initially thought to be core components of the endocytic machinery because of the dominant-negative effects of truncated Epsin proteins on endocytosis in mammalian cells (Chen, 1998; Ford, 2002), their essential role in yeast endocytosis (Wendland, 1999), and their inherent capacity to induce membrane curvature (Ford, 2002) and bind other core components such as Clathrin and AP-2 (Chen, 1998; Owen, 1999; Rosenthal, 1999; Wendland, 1999; Drake, 2000). More recently, however, the identification of Ubiquitin-interacting motifs (UIMs) in Epsins, as well as in other proteins involved in membrane trafficking (Hofmann, 2001), have led to the suggestion (Wendland, 2002) that Epsins belong to a family of cargo-selective adapters that link mono-ubiquitinated cell-surface proteins with the endocytic machinery (Wang, 2004 and references therein).

Ubiquitination of Delta appears necessary for endocyosis of Delta. Two members of the Notch pathway, neuralized and mind bomb, encode E3-ubiquitin-ligases, ubiquitinate Delta and appear to be required for Delta internalization; loss of neuralized or mind bomb function causes excessively high levels of Delta at the cell membrane and blocks Notch signaling. The Drosophila homolog of epsin, liquid facets (lqf), has also been shown to promote Delta internalization (Overstreet, 2003), suggesting lqf may regulate Notch signaling even though the functional ramifications of lqf on Notch pathway activity have not been investigated (Tian, 2004 and references therein).

Delta/Serrate/Lag2 (DSL) ligands must normally be endocytosed in signal-sending cells via the action of Liquid facets to activate Notch on the surface of signal-receiving cells. Surprisingly, however, bulk endocytosis of DSL ligands appears normal in the absence of Lqf. This apparent paradox is resolved by providing evidence that Lqf is unique among adapters that target mono-ubiquitinated cargo proteins for internalization: it allows them to enter a special endocytic pathway that DSL ligands must enter to acquire signaling activity. This requirement can be bypassed by introducing the internalization signal that normally mediates internalization and recycling of the Low Density Lipoprotein (LDL) receptor. On the basis of these results, it is hypothesized that Epsin-mediated endocytosis might be required to allow DSL proteins to be recycled rather than degraded following internalization, possibly to convert them from inactive pro-ligands into active ligands (Wang, 2004).

Cardioblasts are the contractile cells of the heart and coalesce to form the heart tube. liquid facets functions as an inhibitor of cardioblast development in Drosophila. lqf inhibits cardioblast development and promotes the development of fusion-competent myoblasts, suggesting a model in which lqf acts on or in fusion-competent myoblasts to prevent their acquisition of the cardioblast fate. lqf and Notch exhibit essentially identical heart phenotypes, and lqf genetically interacts with the Notch pathway during multiple Notch-dependent events in Drosophila. The link between the Notch pathway and epsin function has been extended to C. elegans, where the C. elegans Lqf ortholog acts in the signaling cell to promote the glp-1/Notch pathway activity during germline development. These results suggest that epsins play a specific, evolutionarily conserved role to promote Notch signaling during animal development and support the idea that they do so by targeting ligands of the Notch pathway for endocytosis (Tian, 2004).

Epsins, including the sole Drosophila Epsin Lqf, contain a series of discrete functional domains that implicate them in Clathrin-mediated endocytosis. These include the N-terminal ENTH domain, which can induce membrane curvature in response to PtdIns(4,5)P₂ binding, as well as binding sites for Clathrin, AP-2 and accessory proteins (e.g. Eps15), and multiple Ubiquitin Interactions Motifs (UIMs). Recent evidence (Mishra, 2002; Wendland, 2002) has suggested that Epsins are members of a class of structurally related proteins that function as cargo selective adapters that target substrate proteins for Clathrin-mediated endocytosis (Wang, 2004).

An absolute, cell-autonomous requirement for Lqf in generating functional DSL ligands has been demonstrated. However, no other role has been detected for Lqf in cell-cell signaling, such as in receiving DSL ligands, or in sending, receiving, or controlling the distribution of other extracellular signals, notably Hg, Wg and Dpp. Furthermore, cells devoid of Lqf activity appear to grow, proliferate and interdigitate in a manner that is indistinguishable from cells devoid of Dl and Ser, the two DSL ligands in Drosophila. These results suggest that Epsin function in Drosophila may be essential solely for the production of active DSL ligands (Wang, 2004).

Surprisingly, no effect of removing Lqf was detected on the steady state accumulation of Dl in endocytic compartments. However, a modest effect on Dl internalization was detected in a sensitized background in which Dl endocytosis was greatly enhance by overexpressing Neuralized, an E3-Ubiquitin ligase that ubiquitinates Dl. Strikingly, high levels of Dl accumulate in endocytic vesicles of such Neur overexpressing cells whether or not they have Lqf, but only cells that have Lqf can signal. It is therefore inferred that Epsin is required for a discrete and apparently small subset of the endocytic events that normally internalize DSL ligands; however, it is this subset that is crucial for generating active DSL signals (Wang, 2004).

The selective requirement for Epsin in sending DSL ligands is reminiscent of that for the Presenilin/gamma-secretase complex in transmembrane cleavage and signal transduction by Notch. Selectivity in the case of the Presenilin/gamma-secretase complex does not reflect a dedicated role in Notch proteolysis, but rather an unusual property of the Notch transduction mechanism, namely that ectodomain shedding activates the pathway by inducing transmembrane proteolysis. Similarly, selectivity in the case of Epsin may reflect an unusual requirement for DSL ligands to signal, and not a dedicated role of Epsin in confering their signaling activity (Wang, 2004).

It is generally thought that Epsins target cargo proteins for endocytosis via mono-ubiquitin internalization signals (Wendland, 2002). The cytosolic domain of Dl was replaced with a random peptide (R+) that contains two Lysines, and the presence of at least one Lysine was shown to be essential for both the endocytosis and signaling activity of the chimeric Dl^R+ ligand. To test the possibility that the presence of Lysine targets Dl^R+, as well as wild-type Dl, for endocytosis by serving as an Ubiquitin acceptor, the cytosolic domain of Dl was replaced with a non-Lysine containing form of Ubiquitin. The resulting Dl^Ubi+ ligand could be endocytosed and had at least partial signaling activity, but not if the Ubiquitin domain contained an additional mutation that blocks its ability to be targeted for endocytosis. Finally, and critically, it was demonstrated that the signaling activity of Dl^R+, like that of wild-type Dl, depends on Lqf. Collectively, these findings implicate mono-ubiquitination as the internalization signal required to target DSL ligands for endocytosis by Epsin (Wang, 2004).

Significantly, bulk endocytosis of the chimeric Dl^R+ ligand, like that of wild-type Dl, appears to be unaffected in cells devoid of Lqf, even though signaling activity is abolished. Hence, it is inferred that Lqf is not the only adapter protein that can target mono-ubiquitinated substrate proteins for endocytosis. Nevertheless, Lqf appears to be unique among all such adapter proteins in its ability to direct internalization of mono-ubiquitinated DSL ligands in a manner that confers signaling activity. It is therefore suggested that Epsin has a dedicated role in directing mono-ubiquitinated cargo proteins into a particular endocytic pathway, one that DSL ligands must enter in order to acquire signaling activity. As is detailed below, it is suggest that Epsin might direct DSL ligands specificially into a recycling pathway (Wang, 2004).

It is notable that substitution of the cytosolic domain of Dl with a peptide carrying the FDNPVY internalization signal from the LDL receptor yields a chimeric Dl^LDL+ ligand that is endocytosed and has signaling activity. However, in this case, Lqf is not essential for signaling. One interpretation of this result is that mono-ubiquitinated DSL ligands are normally targeted for endocytic pathways that preclude their signaling activity, unless they are diverted from entering these pathways by association with Lqf, or by the presence of a heterologous internalization signal such as FDNPVY. In both cases, endocytosis would take place via an alternate pathway compatible with signaling activity (Wang, 2004).

Why must DSL ligands on the surface of signal-sending cells be endocytosed in order to activate Notch on the surface of signal-receiving cells? Two general classes of explanation can be distinguished. In the first, activation of Notch is triggered by early events in the process of DSL endocytosis that occurs while the ligands are still on the cell surface, prior to the pinching off of coated vesicles. In the second, internalization of DSL proteins is a necessary prerequisite for endocytic recycling, which is required for subsequent signaling activity (Wang, 2004).

Most previous models fall into the first class. One such internalization model proposed that DSL/Notch binding creates a physical bridge between the sending and receiving cell that is mechanically stressed by endocytosis of the ligand, causing conformational changes in Notch that elicit either S2 or S3 cleavage. Another model proposed that recruitment of DSL ligands into coated pits increases their local abundance on the cell surface. Both of these models are difficult to reconcile with the finding that Lqf is essential for Dl signaling but not for bulk endocytosis of Dl. This result indicates that DSL endocytosis in signal-sending cells is not sufficient, per se, to activate Notch in signal-receiving cells. Instead, as suggested above, it appears that DSL ligands have to enter, or traffic through, a special Lqf-dependent endocytic pathway to activate Notch (Wang, 2004).

For such internalization models to accommodate these results, it seems necessary to posit that productive interactions between DSL ligands and Notch require a special micro-environment that is associated only with a particular subclass of coated pits or other specializations. Mono-ubiquitinated cargo proteins might be excluded from such structures, unless chaperoned there by Lqf. Thus, only DSL ligands that gain entry, whether via Lqf, or by the targeting mediated by the LDL receptor signal, would be able to activate Notch on the abutting surface of the receiving cell. Furthermore, one would have to posit the existence of accessory molecules that are provided by the sending cell, sequestered in these specializations, and essential for DSL-dependent activation of Notch on the receiving cell, whether by mechanical stress, DSL clustering, or some other means (Wang, 2004).

The second general class of explanation suggests, by the ability of the internalization signal from the LDL receptor to bypasses the requirement for Lqf, that recycling is the key element. In general, mono-ubiquitination acts as a sorting signal in the endosomal system that leads to delivery of membrane proteins to late endosomes and eventually lysosomes. Hence, Epsin-binding to mono-ubiquitinated DSL proteins during endocytosis might allow those DSL proteins to escape degradation by altering their sorting, thus allowing them to enter a recycling pathway. Passage through this pathway would be essential to confer signaling activity (Wang, 2004).

Why might recycling be necessary for DSL ligands to acquire signaling activity? One possibility is that recycling allows DSL ligands to be stripped of the bound ectodomain of Notch so that they can be re-used. Multiple rounds of recycling might then enhance the level of active DSL ligands on the surface of signal-sending cells above a critical threshold necessary to activate Notch transduction in the signal-receiving cell. According to this view, one might expect that massive overexpression of DSL ligands would be able to bypass the requirement for Lqf. However, the results suggest that this is not the case: it is estimated that, in these experiments, overexpressed Dl accumulates on the cell surface at levels up to tenfold higher than peak accumulation of endogenous Dl, yet is unable to rescue DSL signaling activity in cells devoid of Lqf (Wang, 2004).

Alternatively, recycling of nascent DSL proteins may be important to convert inactive 'pro-ligands' into active ligands. Conversion might entail recruitment of DSL proteins into signaling exosomes. However, Dl signaling appears to be unaffected in cells devoid of Hrs, despite impairment in the maturation of early to late endosomes, and in the formation of multi-vesicular bodies from which exosomes might derive. Another possibility is that DSL proteins need to be processed in order to be converted to active ligands, a hypothesis that is consistent with the evidence that Lqf-dependent endocytosis of Dl correlates with a specific proteolytic cleavage of the ligand. Lqf would be required in this scenario to allow DSL ligands to enter a recycling pathway in which the required processing event can occur. The only specificity one needs to invoke in this model is that of Epsin to allow mono-ubiquitinated cargo proteins to gain access to a recycling pathway. The conditions necessary to convert DSL pro-ligands into active signals (e.g., low pH) might exist generally in early endosomes or recycling endosomes (Wang, 2004).  

GENE STRUCTURE

cDNA clone length - 4122 (transcript B)

Bases in 5' UTR - 403

Exons - 9 (transcript B)

Bases in 3' UTR - 1244

PROTEIN STRUCTURE

Amino Acids - 824 (transcript B)

Structural Domains

See InterPro for information on the ENTH (Epsin N-terminal homology) domain and InterPro for information on the Ubiquitin interacting motif.

date revised: 27 December 2004

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.