The EH network

The EH domain is an evolutionary conserved protein-protein interaction domain present in a growing number of proteins from yeast to mammals. Even though the domain was discovered just 5 years ago, a great deal has been learned regarding its three-dimensional structure and binding specificities. Moreover, a number of cellular ligands of the domain have been identified and demonstrated to define a complex network of protein-protein interactions in the eukaryotic cell. Interestingly, many of the EH-containing and EH-binding proteins display characteristics of endocytic "accessory" proteins, suggesting that the principal function of the EH network is to regulate various steps in endocytosis. In addition, recent evidence suggests that the EH network might work as an "integrator" of signals controlling cellular pathways as diverse as endocytosis, nucleocytosolic export, and ultimately cell proliferation.     

Regulation of clathrin coat assembly by Eps15 homology domain-mediated interactions during endocytosis

Clathrin-mediated endocytosis involves a coordinated series of molecular events regulated by interactions among a variety of proteins and lipids through specific domains. One such domain is the Eps15 homology (EH) domain, a highly conserved protein-protein interaction domain present in a number of proteins distributed from yeast to mammals. Several lines of evidence suggest that the yeast EH domain-containing proteins Pan1p, End3p, and Ede1p play important roles during endocytosis. Although genetic and cell-biological studies of these proteins suggested a role for the EH domains in clathrin-mediated endocytosis, it was unclear how they regulate clathrin coat assembly. To explore the role of the EH domain in yeast endocytosis, we mutated those of Pan1p, End3p, or Ede1p, respectively, and examined the effects of single, double, or triple mutation on clathrin coat assembly. We found that mutations of the EH domain caused a defect of cargo internalization and a delay of clathrin coat assembly but had no effect on assembly of the actin patch. We also demonstrated functional redundancy among the EH domains of Pan1p, End3p, and Ede1p for endocytosis. Of interest, the dynamics of several endocytic proteins were differentially affected by various EH domain mutations, suggesting functional diversity of each EH domain.     

EHD2 and the novel EH domain binding protein EHBP1 couple endocytosis to the actin cytoskeleton

Here we identified two novel proteins denoted EH domain protein 2 (EHD2) and EHD2-binding protein 1 (EHBP1) that link clathrin-mediated endocytosis to the actin cytoskeleton. EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1. Disruption of EHD2 or EHBP1 function by small interfering RNA-mediated gene silencing inhibits endocytosis of transferrin into EEA1-positive endosomes as well as GLUT4 endocytosis into cultured adipocytes. EHD2 localizes with cortical actin filaments, whereas EHBP1 contains a putative actin-binding calponin homology domain. High expression of EHD2 or EHBP1 in intact cells mediates extensive actin reorganization. Thus EHD2 appears to connect endocytosis to the actin cytoskeleton through interactions of its N-terminal domain with membranes and its C-terminal EH domain with the novel EHBP1 protein.     

AtEHDs in endocytosis

Endocytosis regulates many important and diverse processes in eukaryotic life. EH domain containing proteins function as regulators of endocytosis through protein-protein interactions. Several interactors of mammalian EHDs were identified, including clathrin machinery components. The four human EHD proteins share high homology at the protein level and possess similar domains, but appear to be involved in different stages of intracellular trafficking. EHD1 regulates recycling through the endocytic recycling compartment (ERC). EHD2 has been found to inhibit internalization in mammalians when overexpressed.We have recently investigated the importance of EH domain containing proteins in plant endocytosis. We were able to show that both of the Arabidopsis EHD homologs, termed AtEHD1 and AtEHD2, play important roles in plant endocytosis. Knockdown of AtEHD1 delayed internalization, and overexpression of AtEHD2 inhibited endocytosis. Thus, the function of plant EHDs is highly homologous to that of mammalian EHDs.Key words: endocytosis, endosome, EH domain, EHD1, EHD2, recycling     

Solution structure of Eps15's third EH domain reveals coincident Phe-Trp and Asn-Pro-Phe binding sites

Eps15 homology (EH) domains interact with proteins involved in endocytosis and signal transduction. EH domains bind to Asn-Pro-Phe (NPF) consensus motifs of target proteins. A few EH domains, such as the third EH domain (EH(3)) of human Eps15, prefer to bind Phe-Trp (FW) sequences. The structure of EH(3) has been solved by nuclear magnetic resonance (NMR) spectroscopy and is the first of an FW- and NPF-binding EH domain. Both FW and NPF sequences bind in the same hydrophobic pocket as shown by heteronuclear chemical shift mapping. EH(3) contains the dual EF-hand fold characteristic of the EH domain family, but it binds calcium with high affinity in the first EF-hand rather than the usual coordination in the second EF-hand. Point mutations were designed based on differences in the EH(3) and the second EH domain (EH(2)) of human Eps15 that alter the affinity of the domains for FW or NPF motif peptides. Peptides that mimic binding sites in the potential EH(3) targets Rab, synaptojanin, and the cation-dependent mannose 6-phosphate receptor were used to explore wild-type and mutant affinities. Characterization of the structure and binding properties of an FW- and NPF-binding EH domain and comparison to an NPF-specific EH domain provide important insights into the mechanisms of EH domain ligand recognition.     

The clathrin adaptor Dab2 recruits EH domain scaffold proteins to regulate integrin β1 endocytosis

Endocytic adaptor proteins facilitate cargo recruitment and clathrin-coated pit nucleation. The prototypical clathrin adaptor AP2 mediates cargo recruitment, maturation, and scission of the pit by binding cargo, clathrin, and accessory proteins, including the Eps-homology (EH) domain proteins Eps15 and intersectin. However, clathrin-mediated endocytosis of some cargoes proceeds efficiently in AP2-depleted cells. We found that Dab2, another endocytic adaptor, also binds to Eps15 and intersectin. Depletion of EH domain proteins altered the number and size of clathrin structures and impaired the endocytosis of the Dab2- and AP2-dependent cargoes, integrin β1 and transferrin receptor, respectively. To test the importance of Dab2 binding to EH domain proteins for endocytosis, we mutated the EH domain-binding sites. This mutant localized to clathrin structures with integrin β1, AP2, and reduced amounts of Eps15. Of interest, although integrin β1 endocytosis was impaired, transferrin receptor internalization was unaffected. Surprisingly, whereas clathrin structures contain both Dab2 and AP2, integrin β1 and transferrin localize in separate pits. These data suggest that Dab2-mediated recruitment of EH domain proteins selectively drives the internalization of the Dab2 cargo, integrin β1. We propose that adaptors may need to be bound to their cargo to regulate EH domain proteins and internalize efficiently.     

The Coiled-Coil Domain of EHD2 Mediates Inhibition of LeEix2 Endocytosis and Signaling

Endocytosis has been suggested to be crucial for the induction of plant immunity in several cases. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and LeEix2. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD2 that are required for its inhibitory activity on endocytosis remained unknown. In this work we demonstrate that the coiled-coil domain of EHD2 is crucial for the ability of EHD2 to inhibit endocytosis in plants, as mutant EHD2 forms lacking the coiled-coil lost the ability to inhibit endocytosis and signaling of LeEix2. The coiled-coil was also required for binding of EHD2 to the LeEix2 receptor. It is therefore probable that binding of EHD2 to the LeEix2 receptor is required for inhibition of LeEix2 internalization. We also show herein that the P-loop of EHD2 is important for EHD2 to function properly. The EH domain of AtEHD2 does not appear to be involved in inhibition of endocytosis. Moreover, AtEHD2 influences actin organization and may exert its inhibitory effect on endocytosis through actin re-distribution. The coiled-coil domain of EHD2 functions in inhibition of endocytosis, while the EH domain does not appear to be involved in inhibition of endocytosis.     

EH proteins

Endocytosis is a protein and lipid-trafficking pathway that occurs in all eukaryotic cells. It involves the internalization of plasma membrane proteins and lipids into the cell and the subsequent degradation of proteins in the lysosome or the recycling of proteins and lipids back to the plasma membrane. Over the past decade, studies in yeast and mammalian cells have revealed endocytosis to be a very complex molecular process that depends on regulated interactions between a variety of proteins and lipids. The Eps15 homology (EH) domain is a conserved, modular protein-interaction domain found in several endocytosis proteins. EH proteins can function as key regulators of endocytosis through their ability to interact with many of the other proteins involved in this process.     

A tubular EHD1-containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane

The Eps15 homology (EH) domain-containing protein, EHD1, has recently been ascribed a role in the recycling of receptors internalized by clathrin-mediated endocytosis. A subset of plasma membrane proteins can undergo internalization by a clathrin-independent pathway regulated by the small GTP-binding protein ADP-ribosylation factor 6 (Arf6). Here, we report that endogenous EHD proteins, as well as transgenic tagged EHD1, are associated with long, membrane-bound tubules containing Arf6. EHD1 appears to induce tubule formation, which requires nucleotide cycling on Arf6 and intact microtubules. Mutations in the N-terminal P-loop domain or deletion of the C-terminal EH domain of EHD1 prevent association of EHD1 with tubules or induction of tubule formation. The EHD1 tubules contain internalized major histocompatibility complex class I (MHC-I) molecules that normally traffic through the Arf6 pathway. Recycling assays show that overexpression of EHD1 enhances MHC-I recycling. These observations suggest an additional function of EHD1 as a tubule-inducing factor in the Arf6 pathway for recycling of plasma membrane proteins internalized by clathrin-independent endocytosis.     

The function of EHD2 in endocytosis and defense signaling is affected by SUMO

Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulates many cellular processes. SUMOylation has been shown to regulate cellular localization and function of a variety of proteins, in some cases affecting nuclear import or export. We have previously characterized two EHDs (EH domain containing proteins) in Arabidospis and showed their involvement in plant endocytosis. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and the leucine rich repeat receptor like protein LeEix2, an effect that requires and intact coiled-coil domain. Inhibition of endocytosis of LeEix2 by EHD2 is effective in inhibiting defense responses mediated by the LeEix2 receptor in response to its ligand EIX. In the present work we demonstrate that SUMOylation of EHD2 appears to be required for EHD2-induced inhibition of LeEix2 endocytosis. Indeed, we found that a mutant form of EHD2, possessing a defective SUMOylation site, has an increased nuclear abundance, can no longer be SUMOylated and is no longer effective in inhibiting LeEix2 endocytosis or defense signaling in response to EIX.     

1H, 13C, and 15N chemical shift assignments for the Eps15-EH2-stonin 2 complex

EH domains are protein–protein interaction domains that function in vesicular trafficking and endocytosis. Here, we report the NMR spectral assignments of the high-affinity complex between the second EH domain of Eps15 and a stonin 2 peptide—providing the basis for the characterization of a two-site binding mode.     

Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors.

The involvement of Ral and its downstream molecules in receptor-mediated endocytosis was examined. Expression of either RalG23V or RalS28N, which are known to be constitutively active and dominantnegative forms, respectively, in A431 cells blocked internalization of epidermal growth factor (EGF). Stable expression of RalG23V or RalS28N in CHO-IR cells also inhibited internalization of insulin. Internalization of EGF and insulin was not affected by full-length RalBP1 which is an effector protein of Ral, but was inhibited by its C-terminal region which binds directly to Ral and POB1. POB1 is a binding protein of RalBP1 and has the Eps15 homology (EH) domain. Deletion mutants of POB1 inhibited internalization of EGF and insulin. However, internalization of transferrin was unaffected by Ral, RalBP1, POB1 and their mutants. Epsin and Eps15 have been reported to be involved in the regulation of endocytosis of the receptors for EGF and transferrin. The EH domain of POB1 bound directly to Epsin and Eps15. Taken together with the observation that EGF and insulin activate Ral, these results suggest that Ral, RalBP1 and POB1 transmit the signal from the receptors to Epsin and Eps15, thereby regulating ligand-dependent receptor-mediated endocytosis.     

The function of the endocytic scaffold protein Pan1p depends on multiple domains

Pan1p is an essential protein of the yeast Saccharomyces cerevisiae that is required for the internalization step of endocytosis and organization of the actin cytoskeleton. Pan1p, which binds several other endocytic proteins, is composed of multiple protein-protein interaction domains including two Eps15 Homology (EH) domains, a coiled-coil domain, an acidic Arp2/3-activating region, and a proline-rich domain. In this study, we have induced high-level expression of various domains of Pan1p in wild-type cells to assess the dominant consequences on viability, endocytosis, and actin organization. We found that the most severe phenotypes, with blocked endocytosis and aggregated actin, required expression of nearly full length Pan1p, and also required the endocytic regulatory protein kinase Prk1p. The central coiled-coil domain was the smallest fragment whose overexpression caused any dominant effects; these effects were more pronounced by inclusion of the second EH domain. Co-overexpressing nonoverlapping amino- and carboxy-terminal fragments did not mimic the effects of the intact protein, whereas fragments that overlapped within the coiled-coil region could. Yeast two-hybrid and in vivo coimmunoprecipitation analyses suggest that Pan1 may form dimers or higher order oligomers. Collectively, our data support a view of Pan1p as a dimeric/oligomeric scaffold whose functions require both the amino- and carboxy-termini, linked by the central region.     

Molecular mechanism of NPF recognition by EH domains

Eps15 homology (EH) domains are protein interaction modules that recognize Asn-Pro-Phe (NPF) motifs in their biological ligands to mediate critical events during endocytosis and signal transduction. To elucidate the structural basis of the EH-NPF interaction, the solution structures of two EH-NPF complexes were solved using NMR spectroscopy. The first complex contains a peptide representing the Hrb C-terminal NPFL motif; the second contains a peptide in which an Arg residue substitutes the C-terminal Leu. The NPF residues are almost completely embedded in a hydrophobic pocket on the EH domain surface and the backbone of NPFX adopts a conformation reminiscent of the Asx-Pro type I beta-turn motif. The residue directly following NPF is crucial for recognition and is required to complete the beta-turn. Five amino acids on the EH surface mediate specific recognition of this residue through hydrophobic and electrostatic contacts. The complexes explain the selectivity of the second EH domain of Eps15 for NPF over DPF motifs and reveal a critical aromatic interaction that provides a conserved anchor for the recognition of FW, WW, SWG and HTF ligands by other EH domains.     

EHD1 Functions in Endosomal Recycling and Confers Salt Tolerance

Endocytosis is a crucial process in all eukaryotic organisms including plants. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. Knock-down of EHD1 was shown to have a delayed recycling phenotype in mammalians. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD1 that are required for its activity have not been characterized. In this work we demonstrate that knock-down of EHD1 causes a delayed recycling phenotype and reduces Brefeldin A sensitivity in Arabidopsis seedlings. The EH domain of EHD1 was found to be crucial for the localization of EHD1 to endosomal structures. Mutant EHD1 lacking the EH domain did not localize to endosomal structures and showed a phenotype similar to that of EHD1 knock-down seedlings. Mutants lacking the coiled-coil domain, however, showed a phenotype similar to wild-type or EHD1 overexpression seedlings. Salinity stress is a major problem in current agriculture. Microarray data demonstrated that salinity stress enhances the expression of EHD1, and this was confirmed by semi quantitative RT-PCR. We demonstrate herein that transgenic plants over expressing EHD1 possess enhanced tolerance to salt stress, a property which also requires an intact EH domain.     

SCAMP1 function in endocytosis

Secretory carrier membrane proteins (SCAMPs) are ubiquitous components of recycling vesicles that shuttle between the plasma membrane, endosomes, and the trans-Golgi complex. SCAMPs contain multiple N-terminal NPF repeats and four highly conserved transmembrane regions. NPF repeats often interact with EH domain proteins that function in budding of transport vesicles from the plasma membrane or the Golgi complex. We now show that the NPF repeats of SCAMP1 bind to two EH domain proteins, intersectin 1, which is involved in endocytic budding at the plasma membrane, and gamma-synergin, which may mediate the budding of vesicles in the trans-Golgi complex. Expression of SCAMP1 lacking the N-terminal NPF repeats potently inhibited transferrin uptake by endocytosis. Our data suggest that one of the functions of SCAMPs is to participate in endocytosis via a mechanism which may involve the recruitment of clathrin coats to the plasma membrane and the trans-Golgi network.     

AtEHDs, novel Arabidopsis EH-domain-containing proteins involved in endocytosis

SUMMARY: Endocytosis is an essential process by which the eukaryotic cell internalizes exogenous material. Studies in yeast and mammalian cells have revealed that endocytosis is a complex molecular process depending on regulated interactions between a variety of proteins and lipids through specific modules. One such module is the Eps15 homology (EH) domain, a conserved modular protein-interaction domain found in several endocytic proteins. The EH-domain-containing proteins function as regulators of endocytosis through their ability to interact with other proteins involved in this process. Here we describe the isolation and characterization of two Arabidopsis EH-domain-containing proteins (AtEHD1 and AtEHD2). We show that the two proteins are involved in endocytosis in plant systems and demonstrate that the Arabidopsis EHD proteins function similarly to mammalian EHDs. Similarly to hEHD2, over-expression of AtEHD2 has an inhibitory effect on endocytosis. While transgenic plants over-expressing AtEHD1 had no detectable phenotype, downregulation of AtEHD1 caused retardation of entry of endocytosed material into plant cells.     

Rab11-FIP2, an adaptor protein connecting cellular components involved in internalization and recycling of epidermal growth factor receptors

Rab11-FIP2 is a member of a newly identified family of Rab11-binding proteins that have been implicated in the function of recycling endosomes. Here we show that Rab11-FIP2 may also be involved with the process of receptor-mediated endocytosis. First we demonstrate that Rab11-FIP2 contains an NPF motif that allows it to bind Reps1, a member of a family of EH domain proteins involved in endocytosis. We also show that Rab11-FIP2 associates with the alpha-adaptin subunit of AP-2 complexes, which are known to recruit receptors into clathrin-coated vesicles. Finally, we find that overexpression of Rab11-FIP2 suppresses the internalization of epidermal growth factor receptors, but not transferrin receptors, through binding sites that promote complex formation with Rab11, Reps1, and alpha-adaptin. These findings suggest that Rab11-FIP2 may participate in the coupling of receptor-mediated endocytosis to the subsequent sorting of receptor-containing vesicles in endosomes.     

The Eps15 homology (EH) domain

The Eps15 homology (EH) domain was originally identified as a motif present in three copies at the NH2-termini of Eps15 and of the related molecule Eps15R. Both of these molecules are substrates for the tyrosine kinase activity of the epidermal growth factor receptor and hence the name 'Eps15 homology' or EH domain [Wong et al. (1994) Oncogene 9, 1591-1597; Wong et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9530-9534; Fazioli et al. (1993) Mol. Cell. Biol. 13, 5814-5828] was derived. The motif was subsequently found in several proteins from yeast to nematode, thus establishing its evolutionary conservation. Initial studies with filter-binding assays and phage-displayed libraries demonstrated its protein:protein interaction abilities and identified specific ligands. Subsequently, structural analyses established the molecular bases of recognition between EH domains and cognate peptides. To date, several EH-containing and EH-binding proteins have been identified, which establish in the cell a network of protein:protein interactions, defined as the EH network. This network coordinates cellular functions connected with endocytosis, actin remodeling and intracellular transduction of signals.