Eps15 Homology Domain Proteins Research Articles

EHD1 promotes CP110 ubiquitination by centriolar satellite delivery of HERC2 to the mother centriole.

Primary cilia are sensory organelles that coordinate diverse signaling pathways, controlling development and homeostasis. Progression beyond the early steps of ciliogenesis requires the removal of a distal end protein, CP110, from the mother centriole, a process mediated by Eps15 Homology Domain protein 1 (EHD1). We show that EHD1 regulates CP110 ubiquitination during ciliogenesis, and identify two E3 ubiquitin ligases, HECT domain and RCC1-like domain 2 (HERC2) and mindbomb homolog 1 (MIB1), that interact with and ubiquitinate CP110. We determined that HERC2 is required for ciliogenesis and localizes to centriolar satellites, which are peripheral aggregates of centriolar proteins known to regulate ciliogenesis. We reveal a role for EHD1 in the transport of centriolar satellites and HERC2 to the mother centriole during ciliogenesis. Taken together, our work showcases a mechanism whereby EHD1 controls centriolar satellite movement to the mother centriole, thus delivering the E3 ubiquitin ligase HERC2 to promote CP110 ubiquitination and degradation.

EHD1 promotes the cancer stem cell (CSC)-like traits of glioma cells via interacting with CD44 and suppressing CD44 degradation.

Plenty of evidence has shown that endocytosis plays a key role in cancer progression; however, its effects in the progression of cancer stem cells (CSCs) are still fragmentary. In the present study, we firstly identified that mammalian Eps15 homology domain protein 1 (EHD1), an endocytic and metastasis-associated gene, was upregulated in the 3D non-adherent spheres derived from glioma cells compared to that in the corresponding parental cells. Further functional experiments revealed that EHD1 knockdown reduced the CSC-like traits of glioma cells, which were evident by the decrease of sphere-formation ability, ALDH1 activity, and CSC markers' expression. Additionally, EHD1 knockdown attenuated the tumor-initiating ability of glioma cells in vivo. Furthermore, it was shown that EHD1 bound to CD44, enhanced CD44 stability, and prevented its total ubiquitination. Indeed, overexpression of CD44 rescued the inhibitory effects of EHD1 knockdown on the CSC-like traits of glioma cells. Finally, through the online dataset analysis, we found that EHD1 indeed exhibited a higher level in glioma tissues relative to that in normal tissues, and a positive correlation with CSC markers' expression in glioma tissues. Notably, EHD1 expression was negatively correlated with the overall survival and relapse-free survival of glioma patients. Thus, this work indicates that EHD1 might be a potent target for glioma progression, especially through breaking the EHD1-CD44 interaction.

Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis.

The endocytic protein EHD1 controls primary ciliogenesis by facilitating fusion of the ciliary vesicle and by removal of CP110 from the mother centriole. EHD3, the closest EHD1 paralog, has a similar regulatory role, but initial evidence suggested that the other two more distal paralogs, EHD2 and EHD4 may be dispensable for ciliogenesis. Herein, we define a novel role for EHD4, but not EHD2, in regulating primary ciliogenesis. To better understand the mechanisms and differential functions of the EHD proteins in ciliogenesis, we first demonstrated a requirement for EHD1 ATP‐binding to promote ciliogenesis. We then identified two sequence motifs that are entirely conserved between EH domains of EHD1, EHD3 and EHD4, but display key amino acid differences within the EHD2 EH domain. Substitution of either P446 or E470 in EHD1 with the aligning S451 or W475 residues from EHD2 was sufficient to prevent rescue of ciliogenesis in EHD1‐depleted cells upon reintroduction of EHD1. Overall, our data enhance the current understanding of the EHD paralogs in ciliogenesis, demonstrate a need for ATP‐binding and identify conserved sequences in the EH domains of EHD1, EHD3 and EHD4 that regulate EHD1 binding to proteins and its ability to rescue ciliogenesis in EHD1‐depleted cells.

A novel EHD1/CD44/Hippo/SP1 positive feedback loop potentiates stemness and metastasis in lung adenocarcinoma.

There is growing evidence that endocytosis plays a pivotal role in cancer metastasis. In this study, we first identified endocytic and metastasis-associated genes (EMGs) and then investigated the biological functions and mechanisms of EMGs. Cancer stem cells (CSCs)-like characteristics were evaluated by tumour limiting dilution assays, three-dimensional (3D) spheroid cancer models. Microarray analysis was used to identify the pathways significantly regulated by mammalian Eps15 homology domain protein 1 (EHD1) knockdown. Mass spectrometry (MS) was performed to identify EHD1-interacting proteins. The function of EHD1 as a regulator of cluster of differentiation 44 (CD44) endocytic recycling and lysosomal degradation was determined by CD44 biotinylation and recycling assays. EHD1 was identified as a significant EMG. Knockdown of EHD1 suppressed CSCs-like characteristics, epithelial-mesenchymal transition (EMT), migration and invasion of lung adenocarcinoma (LUAD) cells by increasing Hippo kinase cascade activation. Conversely, EHD1 overexpression inhibited the Hippo pathway to promote cancer stemness and metastasis. Notably, utilising MS analysis, the CD44 protein was identified as a potential binding partner of EHD1. Furthermore, EHD1 enhanced CD44 recycling and stability. Indeed, silencing of CD44 or disruption of the EHD1/CD44 interaction enhanced Hippo pathway activity and reduced CSCs-like traits, EMT and metastasis. Interestingly, specificity protein 1 (SP1), a known downstream target gene of the Hippo-TEA-domain family members 1 (TEAD1) pathway, was found to directly bind to the EHD1 promoter region and induce its expression. Among clinical specimens, the EHD1 expression level in LUAD tissues of metastatic patients was higher than that of non-metastatic patients. Our findings emphasise that EHD1 might be a potent anti-metastatic target and present a novel regulatory mechanism by which the EHD1/CD44/Hippo/SP1 positive feedback circuit plays pivotal roles in coupling modules of CSCs-like properties and EMT in LUAD. Targeting this loop may serve as a remedy for patients with advanced metastatic LUAD.

Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission.

Upon internalization, receptors are trafficked to sorting endosomes (SE) where they undergo sorting and are then packaged into budding vesicles that undergo fission and transport within the cell. Eps15 Homology Domain Protein 1 (EHD1), the best-characterized member of the Eps15 Homology Domain Protein (EHD) family, has been implicated in catalyzing the fission process that releases endosome-derived vesicles for recycling to the plasma membrane. Indeed, recent studies suggest that upon receptor-mediated internalization, EHD1 is recruited from the cytoplasm to endosomal membranes where it catalyzes vesicular fission. However, the mechanism by which this recruitment occurs remains unknown. Herein, we demonstrate that the EHD1 paralog, EHD4, is required for the recruitment of EHD1 to SE. We show that EHD4 preferentially dimerizes with EHD1, and knock-down of EHD4 expression by siRNA, shRNA or by CRISPR/Cas9 gene-editing leads to impaired EHD1 SE-recruitment and enlarged SE. Moreover, we demonstrate that at least 3 different asparagine-proline-phenylalanine (NPF) motif-containing EHD binding partners, Rabenosyn-5, Syndapin2 and MICAL-L1, are required for the recruitment of EHD1 to SE. Indeed, knock-down of any of these SE-localized EHD interaction partners leads to enlarged SE, presumably due to impaired endosomal fission. Overall, we identify a novel mechanistic role for EHD4 in recruitment of EHD1 to SE, thus positioning EHD4 as an essential component of the EHD1-fission machinery at SE.

Sorting nexin 17 (SNX17) links endosomal sorting to Eps15 homology domain protein 1 (EHD1)–mediated fission machinery

Following endocytosis, receptors that are internalized to sorting endosomes are sorted to different pathways, in part by sorting nexin (SNX) proteins. Notably, SNX17 interacts with a multitude of receptors in a sequence-specific manner to regulate their recycling. However, the mechanisms by which SNX17-labeled vesicles that contain sorted receptors bud and undergo vesicular fission from the sorting endosomes remain elusive. Recent studies suggest that a dynamin-homolog, Eps15 homology domain protein 1, catalyzes fission and releases endosome-derived vesicles for recycling to the plasma membrane. However, the mechanism by which EHD1 is coupled to various receptors and regulates their recycling remains unknown. Here we sought to characterize the mechanism by which EHD1 couples with SNX17 to regulate recycling of SNX17-interacting receptors. We hypothesized that SNX17 couples receptors to the EHD1 fission machinery in mammalian cells. Coimmunoprecipitation experiments and in vitro assays provided evidence that EHD1 and SNX17 directly interact. We also found that inducing internalization of a SNX17 cargo receptor, low-density lipoprotein receptor-related protein 1 (LRP1), led to recruitment of cytoplasmic EHD1 to endosomal membranes. Moreover, surface rendering and quantification of overlap volumes indicated that SNX17 and EHD1 partially colocalize on endosomes and that this overlap further increases upon LRP1 internalization. Additionally, SNX17-containing endosomes were larger in EHD1-depleted cells than in WT cells, suggesting that EHD1 depletion impairs SNX17-mediated endosomal fission. Our findings help clarify our current understanding of endocytic trafficking, providing significant additional insight into the process of endosomal fission and connecting the sorting and fission machineries.

ZmEHD1 Is Required for Kernel Development and Vegetative Growth through Regulating Auxin Homeostasis.

The roles of C-terminal Eps15 homology domain (EHD) proteins in clathrin-mediated endocytosis in plants are poorly understood. Here, we isolated a maize (Zea mays) mutant, designated ehd1, which showed defects in kernel development and vegetative growth. Positional cloning and transgenic analysis revealed that ehd1 encodes an EHD protein. Internalization of the endocytic tracer FM4-64 was substantially reduced in the ehd1 mutant and ZmEHD1 knockout mutants. We further demonstrated that ZmEHD1 and the ZmAP2 σ subunit physically interact at the plasma membrane. Auxin distribution and ZmPIN1a-YFP localization were altered in the ehd1 mutant. Kernel indole-3-acetic acid levels were substantially lower in the ehd1 mutant than in wild-type maize. Exogenous application of 1-naphthaleneacetic acid, but not GA3 or 2-naphthaleneacetic acid, rescued the seed germination and seedling emergency phenotypic defects of ehd1 mutants. Taken together, these results indicate that ZmEHD1 regulates auxin homeostasis by mediating clathrin-mediated endocytosis through its interaction with the ZmAP2 σ subunit, which is crucial for kernel development and vegetative growth of maize.

EHD Proteins Cooperate to Generate Caveolar Clusters and to Maintain Caveolae during Repeated Mechanical Stress

SummaryCaveolae introduce flask-shaped convolutions into the plasma membrane and help to protect the plasma membrane from damage under stretch forces. The protein components that form the bulb of caveolae are increasingly well characterized, but less is known about the contribution of proteins that localize to the constricted neck. Here we make extensive use of multiple CRISPR/Cas9-generated gene knockout and knockin cell lines to investigate the role of Eps15 Homology Domain (EHD) proteins at the neck of caveolae. We show that EHD1, EHD2, and EHD4 are recruited to caveolae. Recruitment of the other EHDs increases markedly when EHD2, which has been previously detected at caveolae, is absent. Construction of knockout cell lines lacking EHDs 1, 2, and 4 confirms this apparent functional redundancy. Two striking sets of phenotypes are observed in EHD1,2,4 knockout cells: (1) the characteristic clustering of caveolae into higher-order assemblies is absent; and (2) when the EHD1,2,4 knockout cells are subjected to prolonged cycles of stretch forces, caveolae are destabilized and the plasma membrane is prone to rupture. Our data identify the first molecular components that act to cluster caveolae into a membrane ultrastructure with the potential to extend stretch-buffering capacity and support a revised model for the function of EHDs at the caveolar neck.

EHD3 Protein Is Required for Tubular Recycling Endosome Stabilization, and an Asparagine-Glutamic Acid Residue Pair within Its Eps15 Homology (EH) Domain Dictates Its Selective Binding to NPF Peptides

An elaborate network of dynamic lipid membranes, termed tubular recycling endosomes (TRE), coordinates the process of endocytic recycling in mammalian cells. The C-terminal Eps15 homology domain (EHD)-containing proteins have been implicated in the bending and fission of TRE, thus regulating endocytic recycling. EHD proteins have an EH domain that interacts with proteins containing an NPF motif. We found that NPF-containing EHD1 interaction partners such as molecules interacting with CasL-like1 (MICAL-L1) and Syndapin2 are essential for TRE biogenesis. Also crucial for TRE biogenesis is the generation of phosphatidic acid, an essential lipid component of TRE that serves as a docking point for MICAL-L1 and Syndapin2. EHD1 and EHD3 have 86% amino acid identity; they homo- and heterodimerize and partially co-localize to TRE. Despite their remarkable identity, they have distinct mechanistic functions. EHD1 induces membrane vesiculation, whereas EHD3 supports TRE biogenesis and/or stabilization by an unknown mechanism. While using phospholipase D inhibitors (which block the conversion of glycerophospholipids to phosphatidic acid) to deplete cellular TRE, we observed that, upon inhibitor washout, there was a rapid and dramatic regeneration of MICAL-L1-marked TRE. Using this "synchronized" TRE biogenesis system, we determined that EHD3 is involved in the stabilization of TRE rather than in their biogenesis. Moreover, we identify the residues Ala-519/Asp-520 of EHD1 and Asn-519/Glu-520 of EHD3 as defining the selectivity of these two paralogs for NPF-containing binding partners, and we present a model to explain the atomic mechanism and provide new insight for their differential roles in vesiculation and tubulation, respectively.

The EHD protein Past1 controls postsynaptic membrane elaboration and synaptic function.

Membranes form elaborate structures that are highly tailored to their specialized cellular functions, yet the mechanisms by which these structures are shaped remain poorly understood. Here, we show that the conserved membrane-remodeling C-terminal Eps15 Homology Domain (EHD) protein Past1 is required for the normal assembly of the subsynaptic muscle membrane reticulum (SSR) at the Drosophila melanogaster larval neuromuscular junction (NMJ). past1 mutants exhibit altered NMJ morphology, decreased synaptic transmission, reduced glutamate receptor levels, and a deficit in synaptic homeostasis. The membrane-remodeling proteins Amphiphysin and Syndapin colocalize with Past1 in distinct SSR subdomains and collapse into Amphiphysin-dependent membrane nodules in the SSR of past1 mutants. Our results suggest a mechanism by which the coordinated actions of multiple lipid-binding proteins lead to the elaboration of increasing layers of the SSR and uncover new roles for an EHD protein at synapses.

Novel functions for the endocytic regulatory proteins MICAL-L1 and EHD1 in mitosis.

During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like-1 (MICAL-L1) and C-terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL-L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi-nucleated cells. We provide evidence that bi-nucleation in MICAL-L1- and EHD1-depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL-L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1-independent function for MICAL-L1 earlier in mitosis. Moreover, we provide evidence that MICAL-L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL-L1 and EHD1 during the cell cycle.

Scratching the surface: actin' and other roles for the C-terminal Eps15 homology domain protein, EHD2.

The C-terminal Eps15 homology domain-containing (EHD) proteins participate in multiple aspects of endocytic membrane trafficking. Of the four mammalian EHD proteins, EHD2 appears to be the most disparate, both in terms of sequence homology, and in subcellular localization/function. Since its initial description as a plasma membrane-associated protein, the precise function of EHD2 has remained enigmatic. Various reports have suggested roles for EHD2 at the plasma membrane, within the endocytic transport system, and even in the nucleus. For example, EHD2 facilitates membrane fusion/repair in muscle cells. Recently the focus has shifted to the role of EHD2 in regulating caveolae. Indeed, EHD2 is highly expressed in tissues rich in caveolae, including fat, muscle and blood vessels. This review highlights cumulative evidence linking EHD2 to actin-rich structures at the plasma membrane, where the plasma membrane-associated phospholipid phosphatidylinositol 4,5-bisphosphate controls EHD2 recruitment. Herein we examine the key pathways where EHD2 might function, and address its potential involvement in these processes.

Differential Roles of C-terminal Eps15 Homology Domain Proteins as Vesiculators and Tubulators of Recycling Endosomes

Endocytic recycling involves the return of membranes and receptors to the plasma membrane following their internalization into the cell. Recycling generally occurs from a series of vesicular and tubular membranes localized to the perinuclear region, collectively known as the endocytic recycling compartment. Within this compartment, receptors are sorted into tubular extensions that later undergo vesiculation, allowing transport vesicles to move along microtubules and return to the cell surface where they ultimately undergo fusion with the plasma membrane. Recent studies have led to the hypothesis that the C-terminal Eps15 homology domain (EHD) ATPase proteins are involved in the vesiculation process. Here, we address the functional roles of the four EHD proteins. We developed a novel semipermeabilized cell system in which addition of purified EHD proteins to reconstitute vesiculation allows us to assess the ability of each protein to vesiculate MICAL-L1-decorated tubular recycling endosomes (TREs). Using this assay, we show that EHD1 vesiculates membranes, consistent with enhanced TRE generation observed upon EHD1 depletion. EHD4 serves a role similar to that of EHD1 in TRE vesiculation, whereas EHD2, despite being capable of vesiculating TREs in the semipermeabilized cells, fails to do so in vivo. Surprisingly, the addition of EHD3 causes tubulation of endocytic membranes in our semipermeabilized cell system, consistent with the lack of tubulation observed upon EHD3 depletion. Our novel vesiculation assay and in vitro electron microscopy analysis, combined with in vivo data, provide evidence that the functions of both EHD1 and EHD4 are primarily in TRE membrane vesiculation, whereas EHD3 is a membrane-tubulating protein.

Cooperation of MICAL-L1, syndapin2, and phosphatidic acid in tubular recycling endosome biogenesis

Endocytic transport necessitates the generation of membrane tubules and their subsequent fission to transport vesicles for sorting of cargo molecules. The endocytic recycling compartment, an array of tubular and vesicular membranes decorated by the Eps15 homology domain protein, EHD1, is responsible for receptor and lipid recycling to the plasma membrane. It has been proposed that EHD dimers bind and bend membranes, thus generating recycling endosome (RE) tubules. However, recent studies show that molecules interacting with CasL-Like1 (MICAL-L1), a second, recently identified RE tubule marker, recruits EHD1 to preexisting tubules. The mechanisms and events supporting the generation of tubular recycling endosomes were unclear. Here, we propose a mechanism for the biogenesis of RE tubules. We demonstrate that MICAL-L1 and the BAR-domain protein syndapin2 bind to phosphatidic acid, which we identify as a novel lipid component of RE. Our studies demonstrate that direct interactions between these two proteins stabilize their association with membranes, allowing for nucleation of tubules by syndapin2. Indeed, the presence of phosphatidic acid in liposomes enhances the ability of syndapin2 to tubulate membranes in vitro. Overall our results highlight a new role for phosphatidic acid in endocytic recycling and provide new insights into the mechanisms by which tubular REs are generated.

EHDs meet the retromer

Retrograde trafficking mediates the transport of endocytic membranes from endosomes to the trans-Golgi network (TGN). Dysregulation of these pathways can result in multiple ailments, including late-onset Alzheimer disease. One of the key retrograde transport regulators, the retromer complex, is tightly controlled by many factors, including the C-terminal Eps15 homology domain (EHD) proteins. This mini-review focuses on recent findings and discusses the regulation of the retromer complex by EHD proteins and the novel EHD1 interaction partner, Rabankyrin-5 (Rank-5).

Intersectin 1 forms complexes with SGIP1 and Reps1 in clathrin-coated pits

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein involved in clathrin-mediated endocytosis, cellular signaling and cytoskeleton rearrangement. ITSN1 gene is located on human chromosome 21 in Down syndrome critical region. Several studies confirmed role of ITSN1 in Down syndrome phenotype. Here we report the identification of novel interconnections in the interaction network of this endocytic adaptor. We show that the membrane-deforming protein SGIP1 ( Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1) and the signaling adaptor Reps1 ( RalBP associated Eps15-homology domain protein) interact with ITSN1 in vivo. Both interactions are mediated by the SH3 domains of ITSN1 and proline-rich motifs of protein partners. Moreover complexes comprising SGIP1, Reps1 and ITSN1 have been identified. We also identified new interactions between SGIP1, Reps1 and the BAR ( Bin/ amphiphysin/ Rvs) domain-containing protein amphiphysin 1. Immunofluorescent data have demonstrated colocalization of ITSN1 with the newly identified protein partners in clathrin-coated pits. These findings expand the role of ITSN1 as a scaffolding molecule bringing together components of endocytic complexes.

EH Domain Proteins Regulate Cardiac Membrane Protein Targeting

Cardiac membrane excitability is tightly regulated by an integrated network of membrane-associated ion channels, transporters, receptors, and signaling molecules. Membrane protein dynamics in health and disease are maintained by a complex ensemble of intracellular targeting, scaffolding, recycling, and degradation pathways. Surprisingly, despite decades of research linking dysfunction in membrane protein trafficking with human cardiovascular disease, essentially nothing is known regarding the molecular identity or function of these intracellular targeting pathways in excitable cardiomyocytes. We sought to discover novel pathways for membrane protein targeting in primary cardiomyocytes. We report the initial characterization of a large family of membrane trafficking proteins in human heart. We used a tissue-wide screen for novel ankyrin-associated trafficking proteins and identified 4 members of a unique Eps15 homology (EH) domain-containing protein family (EHD1, EHD2, EHD3, EHD4) that serve critical roles in endosome-based membrane protein targeting in other cell types. We show that EHD1-4 directly associate with ankyrin, provide the first information on the expression and localization of these molecules in primary cardiomyocytes, and demonstrate that EHD1-4 are coexpressed with ankyrin-B in the myocyte perinuclear region. Notably, the expression of multiple EHD proteins is increased in animal models lacking ankyrin-B, and EHD3-deficient cardiomyocytes display aberrant ankyrin-B localization and selective loss of Na/Ca exchanger expression and function. Finally, we report significant modulation of EHD expression following myocardial infarction, suggesting that these proteins may play a key role in regulating membrane excitability in normal and diseased heart. Our findings identify and characterize a new class of cardiac trafficking proteins, define the first group of proteins associated with the ankyrin-based targeting network, and identify potential new targets to modulate membrane excitability in disease. Notably, these data provide the first link between EHD proteins and a human disease model.

Mechanism for the Selective Interaction of C-terminal Eps15 Homology Domain Proteins with Specific Asn-Pro-Phe-containing Partners

Epidermal growth factor receptor tyrosine kinase substrate 15 (Eps15) homology (EH)-domain proteins can be divided into two classes: those with an N-terminal EH-domain(s), and the C-terminal Eps15 homology domain-containing proteins (EHDs). Whereas many N-terminal EH-domain proteins regulate internalization events, the best characterized C-terminal EHD, EHD1, regulates endocytic recycling. Because EH-domains interact with the tripeptide Asn-Pro-Phe (NPF), it is of critical importance to elucidate the molecular mechanisms that allow EHD1 and its paralogs to interact selectively with a subset of the hundreds of NPF-containing proteins expressed in mammalian cells. Here, we capitalize on our findings that C-terminal EH-domains possess highly positively charged interaction surfaces and that many NPF-containing proteins that interact with C-terminal (but not N-terminal) EH-domains are followed by acidic residues. Using the recently identified EHD1 interaction partner molecule interacting with CasL (MICAL)-Like 1 (MICAL-L1) as a model, we have demonstrated that only the first of its two NPF motifs is required for EHD1 binding. Because only this first NPF is followed by acidic residues, we have utilized glutathione S-transferase pulldowns, two-hybrid analysis, and NMR to demonstrate that the flanking acidic residues "fine tune" the binding affinity to EHD1. Indeed, our NMR solution structure of the EHD1 EH-domain in complex with the MICAL-L1 NPFEEEEED peptide indicates that the first two flanking Glu residues lie in a position favorable to form salt bridges with Lys residues within the EH-domain. Our data provide a novel explanation for the selective interaction of C-terminal EH-domains with specific NPF-containing proteins and allow for the prediction of new interaction partners with C-terminal EHDs.

The Endocytic Recycling Protein EHD2 Interacts with Myoferlin to Regulate Myoblast Fusion

Skeletal muscle is a multinucleated syncytium that develops and is maintained by the fusion of myoblasts to the syncytium. Myoblast fusion involves the regulated coalescence of two apposed membranes. Myoferlin is a membrane-anchored, multiple C2 domain-containing protein that is highly expressed in fusing myoblasts and required for efficient myoblast fusion to myotubes. We found that myoferlin binds directly to the eps15 homology domain protein, EHD2. Members of the EHD family have been previously implicated in endocytosis as well as endocytic recycling, a process where membrane proteins internalized by endocytosis are returned to the plasma membrane. EHD2 binds directly to the second C2 domain of myoferlin, and EHD2 is reduced in myoferlin null myoblasts. In contrast to normal myoblasts, myoferlin null myoblasts accumulate labeled transferrin and have delayed recycling. Introduction of dominant negative EHD2 into myoblasts leads to the sequestration of myoferlin and inhibition of myoblast fusion. The interaction of myoferlin with EHD2 identifies molecular overlap between the endocytic recycling pathway and the machinery that regulates myoblast membrane fusion.

A Role for EHD4 in the Regulation of Early Endosomal Transport

All four of the C-terminal Eps15 homology domain (EHD) proteins have been implicated in the regulation of endocytic trafficking. However, the high level of amino acid sequence identity among these proteins has made it challenging to elucidate the precise function of individual EHD proteins. We demonstrate here with specific peptide antibodies that endogenous EHD4 localizes to Rab5-, early embryonic antigen 1 (EEA1)- and Arf6-containing endosomes and colocalizes with internalized transferrin in the cell periphery. Knock-down of EHD4 expression by both small interfering RNA and short hairpin RNA leads to the generation of enlarged early endosomal structures that contain Rab5 and EEA1 as well as internalized transferrin or major histocompatibility complex class I molecules. In addition, cargo destined for degradation, such as internalized low-density lipoprotein, also accumulates in the enlarged early endosomes in EHD4-depleted cells. Moreover, we have demonstrated that these enlarged early endosomes are enriched in levels of the activated GTP-bound Rab5. Finally, we show that endogenous EHD4 and EHD1 interact in cells, suggesting coordinated involvement in the regulation of receptor transport along the early endosome to endocytic recycling compartment axis. The results presented herein provide evidence that EHD4 is involved in the control of trafficking at the early endosome and regulates exit of cargo toward both the recycling compartment and the late endocytic pathway.