The epsins define a family of proteins that interact with components of the clathrin coat and contain a new protein module

Epsin (epsin 1) is an interacting partner for the EH domain-containing region of Eps15 and has been implicated in conjunction with Eps15 in clathrin-mediated endocytosis. We report here the characterization of a similar protein (epsin 2), which we have cloned from human and rat brain libraries. Epsin 1 and 2 are most similar in their NH(2)-terminal region, which represents a module (epsin NH(2) terminal homology domain, ENTH domain) found in a variety of other proteins of the data base. The multiple DPW motifs, typical of the central region of epsin 1, are only partially conserved in epsin 2. Both proteins, however, interact through this central region with the clathrin adaptor AP-2. In addition, we show here that both epsin 1 and 2 interact with clathrin. The three NPF motifs of the COOH-terminal region of epsin 1 are conserved in the corresponding region of epsin 2, consistent with the binding of both proteins to Eps15. Epsin 2, like epsin 1, is enriched in brain, is present in a brain-derived clathrin-coated vesicle fraction, is concentrated in the peri-Golgi region and at the cell periphery of transfected cells, and partially colocalizes with clathrin. High overexpression of green fluorescent protein-epsin 2 mislocalizes components of the clathrin coat and inhibits clathrin-mediated endocytosis. The epsins define a new protein family implicated in membrane dynamics at the cell surface.     

Eps15 mediates vesicle trafficking from the trans-Golgi network via an interaction with the clathrin adaptor AP-1

Eps15 (EGFR pathway substrate clone 15) is well known for its role in clathrin-coated vesicle formation at the plasma membrane through interactions with other clathrin adaptor proteins such as AP-2. Interestingly, we observed that in addition to its plasma membrane localization, Eps15 is also present at the trans-Golgi network (TGN). Therefore, we predicted that Eps15 might associate with clathrin adaptor proteins at the TGN and thereby mediate the formation of Golgi-derived vesicles. Indeed, we have found that Eps15 and the TGN clathrin adaptor AP-1 coimmunoprecipitate from rat liver Golgi fractions. Furthermore, we have identified a 14-amino acid motif near the AP-2-binding domain of Eps15 that is required for binding to AP-1, but not AP-2. Disruption of the Eps15-AP-1 interaction via siRNA knockdown of AP-1 or expression of mutant Eps15 protein, which lacks a 14-amino acid motif representing the AP-1 binding site of Eps15, significantly reduced the exit of secretory proteins from the TGN. Together, these findings indicate that Eps15 plays an important role in clathrin-coated vesicle formation not only at the plasma membrane but also at the TGN during the secretory process.     

Association and Colocalization of Eps15 with Adaptor Protein-2 and Clathrin

Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-α results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both α-adaptin and clathrin. Upon EGF stimulation, Eps15 and α-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.     

Mapping of Eps15 domains involved in its targeting to clathrin-coated pits

Clathrin-coated pit (CCP) formation occurs as a result of the targeting and assembly of cytosolic coat proteins, mainly the plasma membrane clathrin-associated protein complex (AP-2) and clathrin, to the intracellular face of the plasma membrane. In the present study, the mechanisms by which Eps15, an AP-2-binding protein, is targeted to CCPs was analyzed by following the intracellular localization of Eps15 mutants fused to the green fluorescent protein. Our previous results indicated that the N-terminal Eps15 homology (EH) domains are required for CCP targeting. We now show that EH domains are, however, not sufficient for targeting to CCPs. Similarly, neither the central coiled-coil nor the C-terminal AP-2 binding domains were able to address green fluorescent protein to CCPs. Thus, targeting of Eps15 to CCPs likely results from the collaboration between EH domains and another domain of the protein. An Eps15 mutant lacking the coiled-coil domain localized to CCPs showing that Eps15 dimerization is not strictly required. In contrast, Eps15 mutants lacking all AP-2 binding sites showed a dramatic decrease in plasma membrane staining, showing that AP-2 binding sites, together with EH domains, play an important role in targeting Eps15 into CCPs. Finally, the effect of the Eps15 mutants on clathrin-dependent endocytosis was tested by both immunofluorescence and flow cytometry. The results obtained showed that inhibition of transferrin uptake was observed only with mutants able to interfere with CCP assembly.     

Eps15R and clathrin regulate EphB2‐mediated cell repulsion

Expression of Eph receptors and their ligands, the ephrins, have important functions in boundary formation and morphogenesis in both adult and embryonic tissue. The EphB receptors and ephrinB ligands are transmembrane proteins that are expressed in different cells and their interaction drives cell repulsion. For cell repulsion to occur, trans‐endocytosis of the inter‐cellular receptor‐ligand EphB‐ephrinB complex is required. The molecular mechanism underlying trans‐endocytosis is poorly defined. Here we show that the process is clathrin‐ and Eps15R‐mediated using Co115 colorectal cell lines stably expressing EphB2 and ephrinB1. Cell repulsion in co‐cultures of EphB2‐ and ephrinB1‐expressing cells is significantly reduced by knockdown of Eps15R but not Eps15. A novel interaction motif in Eps15R, DPFxxLDPF, is shown to bind directly to the clathrin terminal domain in vitro. Moreover, the interaction between Eps15R and clathrin is required for EphB2‐mediated cell repulsion as shown in a rescue experiment in the EphB2 co‐culture assay where wild type Eps15R but not the clathrin‐binding mutant rescues cell repulsion. These results provide the first evidence that Eps15R together with clathrin control EphB/ephrinB trans‐endocytosis and thereby cell repulsion.      

Regulation of complex formation of POB1/epsin/adaptor protein complex 2 by mitotic phosphorylation

RalBP1 and POB1, the downstream molecules of small GTP-binding protein Ral, are involved in receptor-mediated endocytosis together with Epsin and Eps15. The regulation of assembly of the complex of these proteins was examined. RalBP1, POB1, Epsin, and Eps15 formed a complex with alpha-adaptin of AP-2 in Chinese hamster ovary cells, but the formation was reduced in mitotic phase. RalBP1, POB1, Epsin, and Eps15 were all phosphorylated in mitotic phase. The phosphorylated forms of POB1 and Epsin were recognized by the antibody MPM2, which is known to detect mitotic phosphoproteins. POB1 and Epsin were phosphorylated by p34(cdc2) kinase in vitro. Their phosphorylation sites (Ser(411) of POB1 and Ser(357) of Epsin) were determined. Phosphorylated Epsin and Epsin(S357D) formed a complex with alpha-adaptin less efficiently than wild type Epsin. Although the EH domain of POB1 bound directly to Epsin, phosphorylation of Epsin inhibited the binding. Furthermore, Epsin(S357D) but not Epsin(S357A) lost the effect of Epsin on the insulin-dependent endocytosis. These results suggest that phosphorylation of Epsin in mitotic phase inhibits receptor-mediated endocytosis by disassembly of its complex with POB1 and alpha-adaptin.     

Eps15 homology domain-NPF motif interactions regulate clathrin coat assembly during synaptic vesicle recycling

Although genetic and biochemical studies suggest a role for Eps15 homology domain containing proteins in clathrin-mediated endocytosis, the specific functions of these proteins have been elusive. Eps15 is found at the growing edges of clathrin-coated pits, leading to the hypothesis that it participates in the formation of coated vesicles. We have evaluated this hypothesis by examining the effect of Eps15 on clathrin assembly. We found that although Eps15 has no intrinsic ability to assemble clathrin, it potently stimulates the ability of the clathrin adaptor protein, AP180, to assemble clathrin at physiological pH. We have also defined the binding sites for Eps15 on squid AP180. These sites contain an NPF motif, and peptides derived from these binding sites inhibit the ability of Eps15 to stimulate clathrin assembly in vitro. Furthermore, when injected into squid giant presynaptic nerve terminals, these peptides inhibit the formation of clathrin-coated pits and coated vesicles during synaptic vesicle endocytosis. This is consistent with the hypothesis that Eps15 regulates clathrin coat assembly in vivo, and indicates that interactions between Eps15 homology domains and NPF motifs are involved in clathrin-coated vesicle formation during synaptic vesicle recycling.     

AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.     

Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells

To assess the contribution of individual endocytic proteins to the assembly of clathrin coated pits, we depleted the clathrin heavy chain and the alpha-adaptin subunit of AP-2 in HeLa-cells using RNA interference. 48 h after transfection with clathrin heavy chain-specific short interfering RNA both, the heavy and light chains were depleted by more than 80%. Residual clathrin was mainly membrane-associated, and an increase in shallow pits was noted. The membrane-association of adaptors, clathrin assembly lymphoid myeloid leukemia protein (CALM), epsin, dynamin, and Eps15 was only moderately affected by the knockdown and all proteins still displayed a punctate staining distribution. Clathrin depletion inhibited the uptake of transferrin but not that of the epidermal growth factor. However, efficient sorting of the epidermal growth factor into hepatocyte growth factor-regulated tyrosine kinase substrate-positive endosomes was impaired. Depletion of alpha-adaptin abolished almost completely the plasma membrane association of clathrin. Binding of Eps15 to membranes was strongly and that of CALM moderately reduced. Whereas the uptake of transferrin was efficiently blocked in alpha-adaptin knockdown cells, the internalization and sorting of the epidermal growth factor was not significantly impaired. Since neither clathrin nor AP-2 is essential for the internalization of EGF, we conclude that it is taken up by an alternative mechanism.     

Tandem arrangement of the clathrin and AP-2 binding domains in amphiphysin 1 and disruption of clathrin coat function by amphiphysin fragments comprising these sites

Amphiphysin 1 and 2 are proteins implicated in the recycling of synaptic vesicles in nerve terminals. They interact with dynamin and synaptojanin via their COOH-terminal SH3 domain, whereas their central regions contain binding sites for clathrin and for the clathrin adaptor AP-2. We have defined here amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin. Overexpression in Chinese hamster ovary cells of an amphiphysin 1 fragment that binds both AP-2 and clathrin resulted in a segregation of clathrin, which acquired a diffuse distribution, from AP-2, which accumulated at patches also positive for Eps15. These effects correlated with a block in clathrin-mediated endocytosis. A fragment selectively interacting with clathrin produced a similar effect. These results can be explained by the binding of amphiphysin to the NH(2)-terminal domain of clathrin and by a competition with the binding of this domain to the beta-subunit of AP-2 and AP180. The interaction of amphiphysin 1 with either clathrin or AP-2 did not prevent its interaction with dynamin, supporting the existence of tertiary complexes between these proteins. Together with previous evidence indicating a direct interaction between amphiphysin and membrane lipids, these findings support a model in which amphiphysin acts as a multifunctional adaptor linking the membrane to coat proteins and coat proteins to dynamin and synaptojanin.     

Ubiquitin-interacting motifs of Epsin are involved in the regulation of insulin-dependent endocytosis

Epsin is a key molecule in receptor-mediated endocytosis. Epsin is phosphorylated and ubiquitinated, and these post-translational modifications are necessary for the regulation of endocytosis. Since human Epsin (hEpsin) has two ubiquitin-interacting motifs (UIMs), we investigated the roles of these UIMs in endocytosis. hEpsin formed a complex with ubiquitinated proteins but did not bind to monoubiquitin. Neither of the two UIMs of hEpsin alone was sufficient to form a complex with ubiquitinated proteins: both UIMs were necessary. Mutations of Asp209 and Asp210 to Ala in UIM (hEpsinDA) abolished the binding activity of hEpsin to ubiquitinated proteins. However, hEpsinDA interacted with Eps15, POB1, and AP-2, which are involved in receptor-mediated endocytosis, as efficiently as wild-type hEpsin. Expression of hEpsinDA in CHO-IR cells affected neither the binding of insulin to nor insulin-dependent autophosphorylation of its receptor. Expression of wild-type hEpsin inhibited the internalization of insulin, whereas that of hEpsinDA did not. These results suggest that the UIM motifs of hEpsin interact with proteins modified with ubiquitin, and that the complex formation is involved in insulin-dependent receptor endocytosis.     

Novel Binding Partners and Differentially Regulated Phosphorylation Sites Clarify Eps8 as a Multi-Functional Adaptor

Eps8 is involved in both cell signalling and receptor trafficking. It is a known phosphorylation substrate for two proteins involved in the fibroblast growth factor receptor (FGFR) signalling pathway: the receptor itself and Src. Here we report a differential proteomic analysis of Eps8 aimed to identify specific FGFR and Src family kinase dependent phosphosites and co-associated phosphodependent binding partners. This study reveals a total of 22 Eps8 pTyr and pSer/Thr phosphorylation sites, including those that are dependent on Src family and FGFR kinase activity. Peptide affinity purification of proteins that bind to a selection of the pTyr phosphosites has identified a range of novel Eps8 binding partners including members of the intracellular vesicle trafficking machinery (clathrin and AP-2), proteins which have been shown to regulate activated receptor trafficking (NBR1 and Vav2), and proteins involved in receptor signalling (IRS4 and Shp2). Collectively this study significantly extends the understanding of Eps8 post-translational modification by regulated phosphorylation, identifies novel Eps8 binding partners implicated in receptor trafficking and signalling, and confirms the functions of Eps8 at the nexus of receptor signalling and vesicular trafficking.     

Direct interaction of the 170 kDa isoform of synaptojanin 1 with clathrin and with the clathrin adaptor AP-2

Synaptojanin 1, a polyphosphoinositide phosphatase, is expressed as two major alternatively spliced isoforms of 145 kDa (SJ145) and 170 kDa (SJ170) [1] [2], which are thought to have pleiotropic roles in endocytosis, signaling and actin function [3] [4] [5]. SJ145 is highly enriched in nerve terminals where it participates in clathrin-dependent synaptic vesicle recycling [1] [5]. SJ170, which differs from SJ145 by the presence of a carboxy-terminal extension, is the predominant isoform in developing neurons and is expressed in a variety of tissues [2]. The carboxy-terminal domain unique to SJ170 was previously shown to bind Eps15 [6], a protein involved in receptor-mediated endocytosis. Here, we show that the same domain also binds clathrin and the clathrin adaptor AP-2. These interactions occur both in vitro and in vivo and are direct. Binding of AP-2 is mediated by the ear domain of its alpha-adaptin subunit and binding of clathrin by the amino-terminal domain of its heavy chain. Overexpression in chinese hamster ovary (CHO) cells of full-length SJ170 or its unique carboxy-terminal region caused mislocalization of Eps15, AP-2 and clathrin, as well as inhibition of clathrin-dependent transferrin uptake. These findings suggest a close association of SJ170 with the clathrin coat and provide new evidence for its physiological role in the regulation of clathrin coat dynamics.     

Clathrin and clathrin-accessory proteins in rat kidney cortex epithelia

Several vectorial transport routes in mammalian cells involve clathrin and associated proteins. In kidney epithelia urine production requires numerous transport processes. However, only little is known about the distribution of clathrin and its associated proteins in this organ in situ. We now report on the presence and distribution of clathrin and its accessory proteins AP1, AP2, Eps15, Epsin, CALM and Clint/EpsinR in the epithelia of the rat kidney cortex using immunoblotting, immunofluorescence and immuno-electron microscopy. Our data show that all investigated proteins are ubiquitously present in rat kidney cortex epithelia, however, with distinct distribution patterns. In the renal corpuscle, podocytes showed the most conspicuous labelling. Clathrin, AP2 and CALM were highly expressed in foot processes, while AP1 was primarily localized in the cell body. In the proximal tubule all proteins were present in dots along the plasma membrane and most conspicuous below the brush border. However, clathrin and AP2 co-localized in vesicle subtypes distinct from those containing clathrin and AP1. In the distal tubule and in the cortical collecting duct all proteins were found in the apex of the cells; however, AP1 and Clint/EpsinR showed additional staining in perinuclear dots. The occurrence and distribution of the investigated proteins in kidney epithelia are discussed with respect to their possible involvement in the functions of the specific nephron segment.Preliminary results were presented at the 28th Annual Meeting of the German Society for Cell Biology, March 16th–19th 2005, Heidelberg, Germany.     

Eps15 Is Recruited to the Plasma Membrane upon Epidermal Growth Factor Receptor Activation and Localizes to Components of the Endocytic Pathway during Receptor Internalization

Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.     

Computational modeling suggests binding-induced expansion of Epsin disordered regions upon association with AP2

Intrinsically disordered regions (IDRs) are prevalent in the eukaryotic proteome. Common functional roles of IDRs include forming flexible linkers or undergoing allosteric folding-upon-binding. Recent studies have suggested an additional functional role for IDRs: generating steric pressure on the plasma membrane during endocytosis, via molecular crowding. However, in order to accomplish useful functions, such crowding needs to be regulated in space (e.g., endocytic hotspots) and time (e.g., during vesicle formation). In this work, we explore binding-induced regulation of IDR steric volume. We simulate the IDRs of two proteins from Clathrin-mediated endocytosis (CME) to see if their conformational spaces are regulated via binding-induced expansion. Using Monte-Carlo computational modeling of excluded volumes, we generate large conformational ensembles (3 million) for the IDRs of Epsin and Eps15 and dock the conformers to the alpha subunit of Adaptor Protein 2 (AP2α), their CME binding partner. Our results show that as more molecules of AP2α are bound, the Epsin-derived ensemble shows a significant increase in global dimensions, measured as the radius of Gyration (R_G) and the end-to-end distance (EED). Unlike Epsin, Eps15-derived conformers that permit AP2α binding at one motif were found to be more likely to accommodate binding of AP2α at other motifs, suggesting a tendency toward co-accessibility of binding motifs. Co-accessibility was not observed for any pair of binding motifs in Epsin. Thus, we speculate that the disordered regions of Epsin and Eps15 perform different roles during CME, with accessibility in Eps15 allowing it to act as a recruiter of AP2α molecules, while binding-induced expansion of the Epsin disordered region could impose steric pressure and remodel the plasma membrane during vesicle formation.     

Epsin 1 is a polyubiquitin-selective clathrin-associated sorting protein

Epsin 1 engages several core components of the endocytic clathrin coat, yet the precise mode of operation of the protein remains controversial. The occurrence of tandem ubiquitin-interacting motifs (UIMs) suggests that epsin could recognize a ubiquitin internalization tag, but the association of epsin with clathrin-coat components or monoubiquitin is reported to be mutually exclusive. Here, we show that endogenous epsin 1 is clearly an integral component of clathrin coats forming at the cell surface and is essentially absent from caveolin-1-containing structures under normal conditions. The UIM region of epsin 1 associates directly with polyubiquitin chains but has extremely poor affinity for monoubiquitin. Polyubiquitin binding is retained when epsin synchronously associates with phosphoinositides, the AP-2 adaptor complex and clathrin. The enrichment of epsin within clathrin-coated vesicles purified from different tissue sources varies and correlates with sorting of multiubiquitinated cargo, and in cultured cells, polyubiquitin, rather than non-conjugable monoubiquitin, promotes rapid internalization. As epsin interacts with eps15, which also contains a UIM region that binds to polyubiquitin, epsin and eps15 appear to be central components of the vertebrate poly/multiubiquitin-sorting endocytic clathrin machinery.     

Epsin 1 is Involved in Recruitment of Ubiquitinated EGF Receptors into Clathrin-Coated Pits

Epsin consists of an epsin NH₂-terminal homology domain that promotes interaction with phospholipids, several AP-2-binding sites, two clathrin-binding sequences and several Eps15 homology domain-binding motifs. Epsin additionally possesses ubiquitin-interacting motifs (UIMs) and has been demonstrated to bind ubiquitinated cargo. We therefore investigated whether epsin promoted clathrin-mediated endocytosis of the ubiquitinated EGF receptor (EGFR). By immunoprecipitation, we found that epsin 1 interacted with ubiquitinated EGFR and that functional UIMs were essential for complex formation. Furthermore, RNA interference-mediated knockdown of epsin 1 was found to inhibit internalization of the EGFR, while having no effect on endocytosis of the transferrin receptor. Additionally, upon knockdown of epsin 1, translocation of the EGFR to central parts of clathrin-coated pits was inhibited. This supports the contention that epsin 1 promotes endocytosis of the ubiquitinated EGFR.