Epsin functions as an adaptor protein which binds to ubiquitinated cargo proteins, clathrin, and phosphoinositides [194, 195]. An ENTH (epsin amino-terminal homology) domain is located at the extreme N-terminus of epsin and is composed of several α-helices that are stabilized by an additional α-helix that is ordered upon PIP2 binding as determined by X-ray crystallography [173, 196]. The ENTH domain of epsin is sufficient to drive curvature of lipids in vitro via utilization of the additional α-helix which inserts into the lipid bilayer suggesting that epsin alone may be sufficient to facilitate curvature of clathrin-coated pits [173]. Additionally, expression of a fluorescently tagged ENTH domain of epsin is targeted to the plasma membrane suggesting that PIP2 binding by the ENTH domain is sufficient for membrane targeting of epsin [197]. The ENTH domain of epsin also interacts with GAPs for small G-proteins, including Cdc42 and Rac [198, 199], suggesting that epsin
may have additional functions outside of its traditional role as a clathrin adaptor protein. The interaction of the ENTH domain of epsin with GAPs for Cdc42 is required for proper cell viability and polarity and actin organization in yeast [199].
C-terminal to the ENTH domain in epsin is three tandem UBDs which are capable of binding to ubiquitin [200]. The UBDs of epsin are necessary for mono-ubiquitination of epsin, although the ubiquitination occurs outside of the UBDs themselves [201, 202]. Recent work has suggested that the UBDs of other clathrin adaptor proteins such as eps15 and Hrs bind intramolecularly to the ubiquitin moiety that is attached to the adaptor protein to inhibit its endocytic function [203]. Furthermore, epsin is basally ubiquitinated and undergoes de- ubiquitination by the DUB, FAM/USP9X, following an increase in intracellular calcium levels (Figure 1.2) [204], thus suggesting that intracellular signaling events may mediate the ability of epsin to recognize ubiquitinated cargo. The UBDs of epsin interact with ubiquitinated EGFR, mediate internalization of the yeast Ste2 receptor and are necessary for functional responses of the epithelial sodium channel (ENaC) [192, 205, 206]. Epsin appears to function in coordination with other adaptor proteins to mediated EGFR and Ste2 internalization and alone to mediate ENaC internalization.
Further evidence for the importance of the UBD domains of epsin in receptor internalization was found in Drosophila melangoster. The development and patterning of photoreceptor cells in the Drosophila compound eye are controlled through signaling by Delta/Notch [207, 208]. Genetic screens searching for proteins involved in eye patterning revealed a role for fat facets (faf) [209] and liquid facets (lqf), the Drosophila homologues of FAM/USP9X and epsin, respectively [210]. Subsequent studies showed that lqf recognizes ubiquitinated Delta and mediates its internalization and signaling [211, 212]. These data
confirm that epsin mediates internalization of cargo via the UBDs which recognize ubiquitin molecules attached to the cargo.
The C-terminus of epsin contains several short peptide motifs that mediate interaction with AP2, clathrin, and EH domain containing proteins. The DPW motifs that are found C- terminal to the UBDs in epsin mediate interaction with the α-adaptin subunit of AP2 [194]. Overexpression of the DPW (aspartic acid/proline/tryptophan) motifs of epsin attenuates internalization of transferrin and EGF presumably by perturbing the network of protein- protein interactions that facilitates general clathrin-mediated endocytosis [194]. A clathrin binding motif which is C-terminal to the DPW motifs, binds to clathrin and exhibits clathrin assembly activity similar to AP2 [213]. By using membrane tethered fluorescently tagged ubiquitin molecules, De Camilli and colleagues suggested that epsin binding to clathrin and ubiquitinated cargo may be mutually exclusive processes [214]; however, whether this phenomenon occurs with bona fide cargo is unclear. Several NPF (Asn-Pro-Phe) motifs which bind to EH domain proteins are found C-terminal to the clathrin binding motif in epsin. Indeed, epsin was first identified by its ability to bind to eps15, which contains EH domains [194].
Eps15/Eps15R
EGFR pathway substrate 15 (Eps15) was originally identified as a substrate of tyrosine phosphorylation upon EGFR activation [215]. Eps15R is a highly related protein that contains additional amino acids inserted between the second and third EH domains. Eps15 and eps15R are encoded by distinct genes, but seems have similar functions [216]. Eps15 and eps15R are composed of three domains. Domain I contains three tandem copies
of an EH domain. EH domains are approximately 100 amino acids in length, are composed of two EF-hand domains connected by a short β-sheet, and bind to NPF motifs in other endocytic adaptor proteins such as epsin [217]. X-ray crystallography of the EH domain of eps15 suggest the EH domain interaction with NPF motifs occurs within a hydrophobic pocket located between two alpha helixes [218]. Domain II contains a coiled-coiled domain that mediates homo- and hetero-dimerization of eps15 and eps15R [219]. Domain III contains DPF motifs that mediate its interaction with the α-adaptin subunit of AP2 [220], UBDs which bind ubiquitinated cargo, and a proline-rich motif which binds to SH3 domains [221]. Similar to epsin, the UBDs of eps15 are necessary for ubiquitination of the adaptor protein which occurs outside of the UBD domains [201]. Interestingly, eps15 and eps15R do not bind clathrin directly, although, the majority of eps15 and eps15R are associated with AP2 and other components of clathrin coated pits in cells [222-224], suggesting a major role in clathrin-mediated endocytosis. Indeed, overexpression of the EH domains of eps15 blocks clathrin-mediated endocytosis and disrupts the distribution of AP2 and clathrin [225, 226]. Based on these observations, overexpression of Domain I of eps15 is often used to examine the role of clathrin-mediated mechanisms in endocytosis of cargo. Further work is needed to determine if eps15 or eps15R directly interact with cargo proteins to mediate internalization.