EHD proteins are associated with tubular and vesicular compartments and interact with specific phospholipids

Blume, Jessica; Halbach, Arndt; Behrendt, Dieter; Paulsson, Mats; Plomann, Markus

doi:10.1016/j.yexcr.2006.10.006

Cited by 67 publications

(92 citation statements)

References 44 publications

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“…A recent report suggests that EHD proteins interact directly with phospholipids (27). To the best of our knowledge, we now provide the first evidence that EH domains are capable of directly interacting with phospholipids.…”

Section: Discussionsupporting

confidence: 51%

“…26). Unlike other mammalian EH domain-containing proteins, this subgroup of four homologous proteins has a single EH domain at its C terminus (27)(28)(29)(30), a central coiled-coil region involved in oligomerization (31)(32)(33), and an N-terminal regulatory region that binds to nucleotides (32,33). Emerging evidence has implicated all four C-terminal EHD proteins in the process of endocytosis (26).…”

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confidence: 99%

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EHD1 and Eps15 Interact with Phosphatidylinositols via Their Eps15 Homology Domains

Naslavsky

Rahajeng

Chenavas

et al. 2007

Journal of Biological Chemistry

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The C-terminal Eps15 homology domain-containing protein, EHD1, regulates the recycling of receptors from the endocytic recycling compartment to the plasma membrane. In cells, EHD1 localizes to tubular and spherical recycling endosomes. To date, the mode by which EHD1 associates with endosomal membranes remains unknown, and it has not been determined whether this interaction is direct or via interacting proteins. Here, we provide evidence demonstrating that EHD1 has the ability to bind directly and preferentially to an array of phospholipids, preferring phosphatidylinositols with a phosphate at position 3. Previous studies have demonstrated that EH domains coordinate calcium binding and interact with proteins containing the tripeptide asparagine-proline-phenylalanine (NPF). Using two-dimensional nuclear magnetic resonance analysis, we now describe a new function for the Eps15 homology (EH) domain of EHD1 and show that it is capable of directly binding phosphatidylinositol moieties. Moreover, we have expanded our studies to include the C-terminal EH domain of EHD4 and the second of the three N-terminal EH domains of Eps15 and demonstrated that phosphatidylinositol binding may be a more general property shared by certain other EH domains. Further studies identified a positively charged lysine residue (Lys-483) localized within the third helix of the EH domain, on the opposite face of the NPF-binding pocket, as being critical for the interaction with the phosphatidylinositols.The internalization of receptors is a critical process for eukaryotic cells. Receptors can be internalized from the plasma membrane by a variety of well described mechanisms, including via clathrin-coated pits, independently of clathrin, and through caveolae (1). Once internalized, the small vesicles containing the internalized cargo fuse with early endosomes (also known as sorting endosomes), and the receptors are then either sent to late endosomes and on to the lysosomal pathway for degradation or recycled back to the plasma membrane, where they may participate in additional rounds of internalization (2). Receptor recycling occurs either directly from the sorting endosomes in a process known as "fast recycling" or indirectly in a process termed "slow" or "regulated" recycling (3). Slow recycling has been better characterized and traverses a complex series of tubular and vesicular membrane structures that emerge from the microtubule-organizing center and is collectively known as the endocytic recycling compartment (3, 4). Despite advances in recent years, the process of recycling is not as well understood as internalization.Among the key regulatory proteins that control endocytic transport and recycling are the Rab family of GTP-binding proteins (5-7). Over 60 different mammalian Rab proteins have been identified thus far, and their highly regulated GTP binding and subsequent hydrolysis lead to recruitment of a wide array of effector proteins that are important for vesicular transport and fusion processes within the endocytic pathways. For exa...

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Section: Discussionsupporting

confidence: 51%

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confidence: 99%

EHD1 and Eps15 Interact with Phosphatidylinositols via Their Eps15 Homology Domains

Naslavsky

Rahajeng

Chenavas

et al. 2007

Journal of Biological Chemistry

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“…The four known carboxyl-terminal EHD proteins are highly related to each other, so it is possible that other EHD proteins participate in myoblasts fusion or in the process of membrane repair in myofibers. Only recently have isoformspecific antibodies been reported for each of the EHD proteins, and these antibodies show that EHD1 and EHD2 are most highly expressed in skeletal muscle, whereas little to no EHD3 and EHD4 is present (46). However, it should be noted that these studies were conducted on skeletal muscle, and the expression pattern of EHD proteins in myoblasts at different stages of differentiation may differ from mature muscle.…”

Section: Discussionmentioning

confidence: 99%

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

Doherty

Demonbreun

Wallace

et al. 2008

Journal of Biological Chemistry

105

137

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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.

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“…There are four vertebrate paralogs [7,8], two plant orthologs and one gene in C. elegans [9] and Drosophila [10]. Vertebrate EHDs show a distinct tissue expression pattern [7,8,[11][12][13] and intracellular localization. EHD1 was localized mainly to the endocytic recycling compartment (ERC) [7] with some localization on the plasma membrane and early endosomes [14,15], while EHD2 was localized to the plasma membrane [16,17].…”

Section: Introductionmentioning

confidence: 99%

“…Total or partial absence of EHD1 or expression of its dominant negative mutants led to attenuated recycling to the plasma membrane [9,15,21,24]. It was recently shown that EHD1 has the ability to bind directly to an array of phospholipids, preferably phosphoinositols with a phosphate at position 3 [11,27]. Several proteins interact with EHD1, including Rabenosyn-5 [28], syndapins I and II [29,30], EHBP1 [23], Rab11-FIP2 (Rab11-family interacting protein 2) [18] and SNAP29 [26,30].…”

Section: Introductionmentioning

confidence: 99%

EHDS are serine phosphoproteins: EHD1 phosphorylation is enhanced by serum stimulation

Fichtman

Ravid

Rapaport

et al. 2008

Cellular and Molecular Biology Letters

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Abbreviations used: AP-2 -adaptor protein complex-2; BSA -bovine serum albumin; CHOChinese hamster ovary; cpm -counts per minute; dFCS -dialyzed FCS; DMEM -Dulbecco modified eagle medium; EHBP1-EH domain binding protein 1; FCS -fetal calf serum; FYVE -phenylalanine-tyrosine-valine-glutamate; IGF-1R -insulin-like growth factor-1 receptor; mDHFR -mouse dihydrofolate reductase; MEM-Alpha -minimal essential medium-alpha; ORF -open reading frame; PBS -phosphate buffered saline; PKC -protein kinase C; PVDFpolyvinidilen difluoride; TLC -thin-layer chromatography; TLE -thin-layer electrophoresis Abstract: Endocytic processes are mediated by multiple protein-protein interacting modules and regulated by phosphorylation and dephosphorylation. The Eps15 homology domain containing protein 1 (EHD1) has been implicated in regulating recycling of proteins, internalized both in clathrin-dependent and clathrin-independent endocytic pathways, from the recycling compartment to the plasma membrane. EHD1 was found in a complex with clathrin, adaptor protein complex-2 (AP-2) and insulin-like growth factor-1 receptor (IGF-1R), and was shown to interact with Rabenosyn-5, SNAP29, EHBP1 (EH domain binding protein 1) and syndapin I and II. In this study, we show that EHD1, like the other human EHDs, undergoes serine-phosphorylation. Our results also indicate that EHD1 is a serum-inducible serine-phosphoprotein and that PKC (protein kinase C) is one of its kinases. In addition, we show that inhibitors of clathrin-mediated endocytosis decrease EHD1 phosphorylation, while inhibitors of caveolinmediated endocytosis do not affect EHD1 phosphorylation. The results of experiments in which inhibitors of endocytosis were employed strongly suggest that EHD1 phosphorylation occurs between early endosomes and the endocytic recycling compartment.

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EHD proteins are associated with tubular and vesicular compartments and interact with specific phospholipids

Cited by 67 publications

References 44 publications

EHD1 and Eps15 Interact with Phosphatidylinositols via Their Eps15 Homology Domains

EHD1 and Eps15 Interact with Phosphatidylinositols via Their Eps15 Homology Domains

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

EHDS are serine phosphoproteins: EHD1 phosphorylation is enhanced by serum stimulation

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