Epsins play a crucial role in cell and organismal physiology as evidenced by their requirement for proper embryo development and cell viability in yeast (Sen et al. 2012). As expected, the functionality of this protein family relies on its ability to interact with multiple cellular factors via an array of binding sites (Fig. 1).
All epsins bear a highly conserved, α-helical epsin N-terminal homology (ENTH) domain followed by a C-terminal unstructured region that collects multiple short protein-binding motifs (Sen et al. 2012 and Fig.1). The ENTH domain interacts with phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid enriched at different regions within the plasma membrane including endocytic sites. It has been shown that this interaction induces the formation of an amphipathic N-terminal alpha-helix in the ENTH domain, which once inserted into the plasma membrane promotes membrane curvature (Ford et al. 2002). This process is thought to contribute to the formation of clathrin-coated pits (Ford et al. 2002).
The C-terminus of mammalian Epns includes several conserved short signature motifs for binding various components of the endocytic machinery (Sen et al. 2012 and Fig.1). For example, this region displays two or three ubiquitin interacting motifs (UIMs) that bind ubiquitinated transmembrane proteins to be internalized. Two clathrin-binding motifs (CBM) that recognize the terminal domain of clathrin heavy chain flank a group of 3–8 DP[W/F] (aspartate-proline-tryptophan/phenylalanine) repeats that interact with the clathrin-associated adaptor complex AP2. In addition, Epns also bear three NPF (asparagine-proline-phenylalanine) tripeptide repeats able to bind the Eps15-homology (EH) domain of proteins like eps15, intersectin, and POB1 (Sen et al. 2012).
Role of Epsin in Endocytosis
Epns play a dual role in endocytosis, as endocytic adaptors and accessory proteins (Fig. 1).
Epns as endocytic adaptors: The presence of conserved UIMs confers the Epn family its ability to interact with ubiquitinated transmembrane proteins and to promote their inclusion in endocytic vesicles as cargoes.
Among the multiple cargoes that epsins contribute to (but are not essential for) their internalization, we find the receptor-bound influenza virus (Chen and Zhuang 2008), dopamine transporter in neurons (Sorkina et al. 2006), and ubiquitinated G protein-coupled receptor protease-activated receptor 1 (PAR1) (Chen et al. 2011). Further, the fly epsin homolog Lqf is involved in promoting receptor-mediated endocytosis of larval serum proteins into hemolymph fat body cells (Csikós et al. 2009).
VEGFR2/3: These proteins are members of the broad receptor tyrosine kinase (RTK) group and are highly relevant to the process of tumorigenesis due to their central role in angiogenesis. Epsin-1 and epsin-2 were shown to be involved in VEGFR2/VEGFR3 internalization in an UIM-dependent manner. Indeed, lack of epsin function led to deficiencies in VEGFR degradation, hyper-VEGF signaling, and abnormal/leaky blood vessel formation (Pasula et al. 2012). Further, administration of an epsin UIM-peptide interfered Epn function in VEGF signaling regulation impairing tumor growth.
Other RTKs, notably insulin receptor (Sugiyama et al. 2005) and epidermal growth factor receptor (EGFR), are also targeted for endocytosis by epsins; however, it is not completely clear if this responsibility is shared with other endocytic adaptors (e.g., Eps15).
EGFR is a member of the ErbB family (erythroblastic leukemia viral oncogene homolog) that includes ErbB1 (EGFR), ErbB2, ErbB3, and ErbB4. Members of this protein family, particularly ErbB1, have been the focus of extensive research due to their role in cancer. Importantly, impaired internalization of EGFR (e.g., by mutations in its cytoplasmic domain) is known to conduce to receptor hyper-signaling and malignant transformation. Following ligand binding to the receptor, the ubiquitin ligase Cbl is known to initiate mono-, multi-, and polyubiquitination, which in turn allows epsin and eps15 recognition via their UIMs (Sen et al. 2012). To what extent this process is solely clathrin-dependent or not is still under debate. The possibility of involvement of other ubiquitin ligases and different posttranslational modifications in EGFR internalization has been also proposed (Goh et al. 2010).
Recently, epsin has been also shown to be responsible for endocytosis and turnover of another member of this receptor family, ErbB3 (Szymanska et al. 2016).
Epithelial sodium channel (ENaC): This channel is primarily found in epithelial cells from the lung, kidney, colon, sweat ducts, and salivary glands. ENaC (made up of α-, β-, and γ-, or δ-subunits) maintains electrolyte balance and fluid movement across the epithelia. Clathrin-mediated internalization of ENaC is initiated by action of the ubiquitin ligase neural precursor cell expressed, developmentally downregulated protein 4–2 (Nedd4–2). In fact, the lack of interaction between ENaC and Nedd4–2 causes Liddle’s syndrome, a pathology characterized by increased channel activity leading to hypertension.
Epsin has been found co-immunoprecipitated with ENaC in an UIM-dependent manner. Furthermore, overexpression of Nedd4–2 or epsin decreased ENaC-dependent currents, suggesting that epsin is involved in the removal from the plasma membrane of the ubiquitinated sodium channel.
Interestingly, the fly potassium-channel Ether-à-go-go was proposed to be a membrane protein able to interact with epsin and, therefore, a prime candidate to also be specific cargo of this endocytic adaptor family.
Notch ligands: The Notch ligand complex is a critical component of a signaling circuit required for embryo development. This signaling pathway is activated by epsins through endocytosis of ubiquitinated notch ligands, such as Delta, Serrate, and Lag2 (see the “Role of Epsin in Signaling” section below).
Epsin as an Accessory Protein
In addition to directly interact with ubiquitinated cargo, epsin-1 contribute to the assembly of the endocytic network by interacting with multiple elements of the endocytosis machinery, including clathrin and eps15 (Fig. 1). Further, Sandra Schmid’s group (UT Southwestern) demonstrated that epsin (along with CALM and SNX9) fulfills an endocytic checkpoint function (Mettlen et al. 2009). The checkpoint functionality relies on protein-protein interactions to “sense” the reaching of critical mass of cargo, endocytic components, and other factors and consequently allows or aborts clathrin-coated pit maturation (Mettlen et al. 2009). This finding has been confirmed and further developed by others (Henry et al. 2012).
Role of Epsin in Signaling
Endocytosis is a known modulator of cell signaling. Internalization of ligand-bound receptors from the plasma membrane followed by routing to the lysosome for degradation is a classical mechanism for signaling termination or attenuation. Epsin participates of this regulatory mechanism, particularly targeting activated RTKs such as EGFR, VEGFR2/VEGFR3 and insulin receptor. However, it is known that internalization does not always equate with signaling inactivation. Indeed, many occupied receptors (including EGFR) initiate novel signaling events once they reach the endosomal compartments (signaling endosome hypothesis). In these cases, endocytosis contributes to the initiation of a signaling event at endosomes. Further, internalization can constitute the essential step of signaling activation. In fact, epsin is the key player necessary for internalization-mediated activation of the juxtacrine Notch signaling pathway (see below).
The epsin family of endocytic adaptors can also directly interact with signaling modulators of RhoGTPases leading to Cdc42/Rac activation (see below). Interestingly, epsin has recently been reported to bind the signaling molecule disheveled segment polarity protein 2 (DVL2), opening the possibility that these endocytic adaptors play a role in the regulation of the crucial Wnt developmental pathway (Chang et al. 2015).
Epsin Plays a Key Role in Notch Signaling Activation
Upon Notch binding, ligands Delta, Serrate, and Lag2 undergo ubiquitination catalyzed by the mind bomb and neuralized ubiquitin ligases. The ubiquitinated proteins are then recognized by epsins and retained in nascent endocytic sites. Epn-mediated internalization of these Notch-bound ligands is thought to induce a mechanical deformation in Notch (on the neighboring cell) that exposes protein sequences susceptible to enzymatic cleavage. The proteolytic release of a cytosolic Notch fragment and its translocation into the nucleus is believed to lead to changes in gene expression that amounts to the cellular signal transduction response to Notch activation.
In agreement with its central role in this pathway activation, epsins are required for embryogenesis and development. Indeed, epsin mutations in flies and worms led to abnormalities in germline and heart development due to defects in Notch signaling. Similarly, double epsin-1/epsin-2 knockout in mice caused embryonic lethality due to abnormalities in extra-embryonic structures and defects in cardiovascular development, somitogenesis, and neural tube differentiation, all these phenotypes being compatible with Notch signaling impairment. These studies suggest that although epsin is dispensable for general endocytosis, it may be critical for cargo-specific signaling functions.
Epsins Contribute to RhoGTPase Signaling and Actin Cytoskeleton Reorganization
In addition to their endocytic role, epsins are involved in the regulation of the activation of the RhoGTPases Cdc42 and Rac1 (Aguilar et al. 2006). From a mechanistic point of view, the epsin’s ENTH domain was shown to bind and inhibit GTPase activating proteins (GAPs) for RhoGTPases (Aguilar et al. 2006). This interaction has been proposed to suppress the GAP-induced inhibition and in that way to promote RhoGTPase signaling activation. It should be noted that this activity is analogous to the one described for the epsin-interacting protein intersectin that sequesters/inhibits a Cdc42 GAP and CdGAP and displays GEF activity towards Cdc42. Since RhoGTPases are central modulators of cell division, membrane trafficking, and actin and microtubule cytoskeleton rearrangements, it is not surprising that these processes are affected in epsin-deficient cells.
In yeast the ENTH domain is necessary and sufficient to sustain cell viability in epsin-deficient cells (Aguilar et al. 2006). As expected, ENTH domain mutations affecting GAP recognition (ENTHY100R and ENTHT104D) led to defects in Cdc42 activation, reduced viability, and actin cytoskeleton depolarization (Aguilar et al. 2006).
Epn deficiency has been also linked to actin cytoskeleton abnormalities in amoeba (Brady et al. 2010) and mammalian (Messa et al. 2014) cells. The observed phenotypes may reflect lack of interaction with the actin-binding protein HIP1, instead of RhoGTPases abnormalities.
Interestingly, yeast epsin Ent2 has also been found to have physical and genetic interactions with the only Saccharomyces cerevisiae Cdc42 GEF.
In addition, the ENTH domain of Ent2 bears an additional activity in cell division signaling pathways (Mukherjee et al. 2009) that is dependent on its ability to interact with one of the yeast Cdc42 GAPs: bud emergence 3 (Bem3). Bem3 is also one of the key players in the assembly of the septin cytoskeleton, a family of scaffolding proteins essential for proper cell division. In fact, overexpression of the ENTH domain of Ent2, but not of Ent1, induces severe abnormalities in septin organization (Mukherjee et al. 2009) and cell division. Indeed, a role for epsins in cell division has also been observed in mammalian cells (Messa et al. 2014), although it is unclear whether RhoGAP are involved.
It is, nevertheless, possible that the endocytic protein epsin is involved in RhoGTPase activation, probably coordinating signaling and endocytosis in time and space.
Importantly, this role of epsin in RhoGTPase signaling is conserved in mammals. Specifically, epsins were found to bind the Cdc42/Rac1 GAP and Ral effector, RalBP1 (Ral binding protein 1) (Coon et al. 2010) and to be essential for cell migration. Indeed, epsin knockdown led to Rac1 and Cdc42 inactivation and impairment of cell migration/invasion.
Interestingly, epsin-3 is selectively expressed in migratory keratinocytes, and epn upregulation has been reported in invasive skin, breast, and lung cancers. Therefore, it is possible that epsin upregulation contributes to enhance cancer cell invasion in vivo by Rho GTPase (Rac1) and Notch signaling hyperactivation and it constitutes an interesting possibility to be investigated.
The epsins constitute a multifaceted protein family that, besides its classical role in cargo-recognition and endocytic network stabilization, plays an important role in cell signaling. Epsin is involved in the activation of the Notch pathway, and it affects RhoGTPase signaling. Therefore, it is not surprising that the epsin family is required for cell viability (yeast), proper embryo development (worms, flies, and mice), as well as establishment of cell polarity and functions associated with it (e.g., cytokinesis, cell migration, and invasion). Unlike other endocytic proteins, the epsins (particularly epsin-3) are found to be upregulated in cancer cells. Although a direct link to carcinogenesis remains to be established, the role of epsins in promoting cancer invasion is a promising direction to be explored.
I thank the members of the Aguilar Lab for input. I apologize to all authors whose original contributions we could not cite due to space limitations. For more complete listings, please refer to the cited reviews and references therein. The Aguilar Lab is supported by grants from the National Science Foundation MCB-1021377 and by the Center for Science of Information (CSoI), an NSF Science and Technology Center, under grant agreement CCF-0939370.
- Brady RJ, Damer CK, Heuser JE, O’Halloran TJ. Regulation of Hip1r by epsin controls the temporal and spatial coupling of actin filaments to clathrin-coated pits. J Cell Sci. 1 Nov 2010;123(Pt 21):3652–3661.Google Scholar
- Chang B, Tessneer KL, McManus J, Liu X, Hahn S, Pasula S, Wu H, Song H, Chen Y, Cai X, Dong Y, Brophy ML, Rahman R, Ma JX, Xia L, Chen H. Epsin is required for Dishevelled stability and Wnt signalling activation in colon cancer development. Nat Commun. 16 Mar 2015;6:6380. doi: 10.1038/ncomms7380.Google Scholar
- Chen B, Dores MR, Grimsey N, Canto I, Barker BL, Trejo J. AP-2 and epsin-1 mediate protease-activated receptor-1 internalization via phosphorylation- and ubiquitination-dependent sorting signals. J Biol Chem. 2011;286(47):40760–70.Google Scholar
- Henry AG, Hislop JN, Grove J, Thorn K, Marsh M, von Zastrow M. Regulation of endocytic clathrin dynamics by cargo ubiquitination. Dev Cell. 11 Sep 2012;23(3):519–532.Google Scholar
- Kang YL, Yochem J, Bell L, Sorensen EB, Chen L, Conner SD. Caenorhabditis elegans reveals a FxNPxY-independent low-density lipoprotein receptor internalization mechanism mediated by epsin1. Mol Biol Cell. Feb 2013;24(3):308–318.Google Scholar
- Liu X, Pasula S, Song H, Tessneer KL, Dong Y, Hahn S, Yago T, Brophy ML, Chang B, Cai X, Wu H, McManus J, Ichise H, Georgescu C, Wren JD, Griffin C, Xia L, Srinivasan RS, Chen H. Temporal and spatial regulation of epsin abundance and VEGFR3 signaling are required for lymphatic valve formation and function. Sci Signal. 14 Oct 2014;7(347):ra97.Google Scholar
- Meloty-Kapella L, Shergill B, Kuon J, Botvinick E, Weinmaster G. Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins, and actin. Dev Cell. 12 Jan 2012;22(6):1299–1312.Google Scholar
- Messa M, Fernández-Busnadiego R, Sun EW, Chen H, Czapla H, Wrasman K, Wu Y, Ko G, Ross T, Wendland B, De Camilli P. Epsin deficiency impairs endocytosis by stalling the actin-dependent invagination of endocytic clathrin-coated pits. Elife. 13 Aug 2014;3:e03311. 10.7554/eLife.03311.Google Scholar
- Pasula S, Cai X, Dong Y, Messa M, McManus J, Chang B, Liu X, Zhu H, Mansat RS, Yoon SJ, Hahn S, Keeling J, Saunders D, Ko G, Knight J, Newton G, Luscinskas F, Sun X, Towner R, Lupu F, Xia L, Cremona O, De Camilli P, Min W, Chen H. Endothelial epsin deficiency decreases tumor growth by enhancing VEGF signaling. J Clin Invest. Dec 2012;122(12):4424–4438.Google Scholar
- Sen A, Madhivanan K, Mukherjee D, Aguilar RC. The epsin protein family: coordinators of endocytosis and signaling. Biomol Concepts. Apr 2012;3(2):117–126.Google Scholar
- Sorrentino V, Nelson JK, Maspero E, Marques AR, Scheer L, Polo S, Zelcer N. The LXR-IDOL axis defines a clathrin-, caveolae-, and dynamin-independent endocytic route for LDLR internalization and lysosomal degradation. J Lipid Res. Aug 2013;54(8):2174–2184.Google Scholar
- Szymanska M, Fosdahl AM, Raiborg C, Dietrich M, Liestøl K, Stang E, Bertelsen V. Interaction with epsin 1 regulates the constitutive clathrin-dependent internalization of ErbB3. Biochim Biophys Acta. Jun 2016;1863(6 Pt A):1179–1188.Google Scholar