EH domain proteins regulate cardiac membrane protein targeting.
ABSTRACT: 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.
Project description:Eps15 homology domain (EHD) 1 enables membrane recycling by controlling the exit of internalized molecules from the endocytic recycling compartment (ERC) en route to the plasma membrane, similar to the role described for Rab11. However, no physical or functional connection between Rab11 and EHD-family proteins has been demonstrated yet, and the mode by which they coordinate their regulatory activity remains unknown. Here, we demonstrate that EHD1 and EHD3 (the closest EHD1 paralog), bind to the Rab11-effector Rab11-FIP2 via EH-NPF interactions. The EHD/Rab11-FIP2 associations are affected by the ability of the EHD proteins to bind nucleotides, and Rab11-FIP2 is recruited to EHD-containing membranes. These results are consistent with a coordinated role for EHD1 and Rab11-FIP2 in regulating exit from the ERC. However, because no function has been attributed to EHD3, the significance of its interaction with Rab11-FIP2 remained unclear. Surprisingly, loss of EHD3 expression prevented the delivery of internalized transferrin and early endosomal proteins to the ERC, an effect differing from that described upon EHD1 knockdown. Moreover, the subcellular localization of Rab11-FIP2 and endogenous Rab11 were altered upon EHD3 knockdown, with both proteins absent from the ERC and retained in the cell periphery. The results presented herein promote a coordinated role for EHD proteins and Rab11-FIP2 in mediating endocytic recycling and provide evidence for the function of EHD3 in early endosome to ERC transport.
Project description:Electrical and structural remodeling during the progression of cardiovascular disease is associated with adverse outcomes subjecting affected patients to overt heart failure (HF) and/or sudden death. Dysfunction in integral membrane protein trafficking has long been linked with maladaptive electrical remodeling. However, little is known regarding the molecular identity or function of these intracellular targeting pathways in the heart. Eps15 homology domain-containing (EHD) gene products (EHD1-4) are polypeptides linked with endosomal trafficking, membrane protein recycling, and lipid homeostasis in a wide variety of cell types. EHD3 was recently established as a critical mediator of membrane protein trafficking in the heart. Here, we investigate the potential link between EHD3 function and heart disease. Using four different HF models including ischemic rat heart, pressure overloaded mouse heart, chronic pacing-induced canine heart, and non-ischemic failing human myocardium we provide the first evidence that EHD3 levels are consistently increased in HF. Notably, the expression of the Na/Ca exchanger (NCX1), targeted by EHD3 in heart is similarly elevated in HF. Finally, we identify a molecular pathway for EHD3 regulation in heart failure downstream of reactive oxygen species and angiotensin II signaling. Together, our new data identify EHD3 as a previously unrecognized component of the cardiac remodeling pathway.
Project description:Cardiac function is dependent on the coordinate activities of membrane ion channels, transporters, pumps, and hormone receptors to tune the membrane electrochemical gradient dynamically in response to acute and chronic stress. Although our knowledge of membrane proteins has rapidly advanced during the past decade, our understanding of the subcellular pathways governing the trafficking and localization of integral membrane proteins is limited and essentially unstudied in vivo. In the heart, to our knowledge, there are no in vivo mechanistic studies that directly link endosome-based machinery with cardiac physiology.To define the in vivo roles of endosome-based cellular machinery for cardiac membrane protein trafficking, myocyte excitability, and cardiac physiology.We identify the endosome-based Eps15 homology domain 3 (EHD3) pathway as essential for cardiac physiology. EHD3-deficient hearts display structural and functional defects including bradycardia and rate variability, conduction block, and blunted response to adrenergic stimulation. Mechanistically, EHD3 is critical for membrane protein trafficking, because EHD3-deficient myocytes display reduced expression/localization of Na/Ca exchanger and L-type Ca channel type 1.2 with a parallel reduction in Na/Ca exchanger-mediated membrane current and Cav1.2-mediated membrane current. Functionally, EHD3-deficient myocytes show increased sarcoplasmic reticulum [Ca], increased spark frequency, and reduced expression/localization of ankyrin-B, a binding partner for EHD3 and Na/Ca exchanger. Finally, we show that in vivo EHD3-deficient defects are attributable to cardiac-specific roles of EHD3 because mice with cardiac-selective EHD3 deficiency demonstrate both structural and electric phenotypes.These data provide new insight into the critical role of endosome-based pathways in membrane protein targeting and cardiac physiology. EHD3 is a critical component of protein trafficking in heart and is essential for the proper membrane targeting of select cellular proteins that maintain excitability.
Project description:Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11-Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11-Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby EHD1 and EHD3 reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth.
Project description:Recycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ). We have previously shown high expression of the endocytic recycling regulator Eps15 homology domain-containing (EHD)1 proteinin the Torpedo californica electric organ, a model tissue for investigating a cholinergic synapse. In this study, we investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1-/- NMJs.Localization of the candidate NMJ protein EHD1 was assessed by confocal microscopy analysis of whole-mount mouse skeletal muscle fibers after direct gene transfer and immunolabeling. The potential function of EHD1 was assessed by specific force measurement and α-bungarotoxin-based endplate morphology mapping in EHD1-/- mouse skeletal muscle.Endogenous EHD1 localized to primary synaptic clefts of murine NMJ, and this localization was confirmed by expression of recombinant green fluorescent protein labeled-EHD1 in murine skeletal muscle in vivo. EHD1-/- mouse skeletal muscle had normal histology and NMJ morphology, and normal specific force generation during muscle contraction. The EHD 1-4 proteins showed differential localization in skeletal muscle: EHD2 to muscle vasculature, EHD3 to perisynaptic regions, and EHD4 to perinuclear regions and to primary synaptic clefts, but at lower levels than EHD1. Additionally, specific antibodies raised against mammalian EHD1-4 recognized proteins of the expected mass in the T. californica electric organ. Finally, we found that EHD4 expression was more abundant in EHD1-/- mouse skeletal muscle than in wild-type skeletal muscle.EHD1 and EHD4 localize to the primary synaptic clefts of the NMJ. Lack of obvious defects in NMJ structure and muscle function in EHD1-/- muscle may be due to functional compensation by other EHD paralogs.
Project description:The four mammalian C-terminal Eps15 homology domain-containing proteins (EHD1-EHD4) play pivotal roles in endocytic membrane trafficking. While EHD1, EHD3 and EHD4 associate with intracellular tubular/vesicular membranes, EHD2 localizes to the inner leaflet of the plasma membrane. Currently, little is known about the regulation of EHD2. Thus, we sought to define the factors responsible for EHD2's association with the plasma membrane. The subcellular localization of endogenous EHD2 was examined in HeLa cells using confocal microscopy. Although EHD partner proteins typically mediate EHD membrane recruitment, EHD2 was targeted to the plasma membrane independent of two well-characterized binding proteins, syndapin2 and EHBP1. Additionally, the EH domain of EHD2, which facilitates canonical EHD protein interactions, was not required to direct overexpressed EHD2 to the cell surface. On the other hand, several lines of evidence indicate that the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) plays a crucial role in regulating EHD2 subcellular localization. Pharmacologic perturbation of PIP2 metabolism altered PIP2 plasma membrane distribution (as assessed by confocal microscopy), and caused EHD2 to redistribute away from the plasma membrane. Furthermore, overexpressed EHD2 localized to PIP2-enriched vacuoles generated by active Arf6. Finally, we show that although cytochalasin D caused actin microfilaments to collapse, EHD2 was nevertheless maintained at the plasma membrane. Intriguingly, cytochalasin D induced relocalization of both PIP2 and EHD2 to actin aggregates, supporting a role of PIP2 in controlling EHD2 subcellular localization. Altogether, these studies emphasize the significance of membrane lipid composition for EHD2 subcellular distribution and offer new insights into the regulation of this important endocytic protein.
Project description:Axon growth is regulated by many proteins, including adhesion molecules, which need to be trafficked correctly to axons. The adhesion molecule L1/neuron-glia cell adhesion molecule (NgCAM) travels to axons via an endocytosis-dependent pathway (transcytosis), traversing somatodendritic endosomes. The Eps15 homology domain (EHD) family proteins (EHD1-EHD4) play important roles in endosomal recycling and possibly in endocytosis. We investigated whether EHD1 regulates L1/NgCAM trafficking in neurons. Both short hairpin-mediated downregulation and overexpression of EHD1 led to dendritic mistargeting of NgCAM. Downregulation of EHD1 showed increased endosomal accumulation of NgCAM, whereas, surprisingly, overexpression of EHD1 led to impairment of L1/NgCAM internalization in neurons but not in fibroblasts. Transferrin internalization, however, was unaffected. At longer overexpression times of EHD1, NgCAM endocytosis returned to normal, suggesting rapid upregulation of compensatory endocytic pathways. EHD1 is capable of hetero-oligomerization, and an endogenous complex of EHD1 and EHD4 was identified previously. We therefore tested whether short-term overexpression of other EHD family members showed a similar endocytosis defect. Expression of EHD4, but not of EHD3, also caused a defect in L1/NgCAM endocytosis. Oligomerization of EHD1 was required to cause NgCAM endocytosis defects, and simultaneous expression of EHD1 and EHD4 rescued NgCAM endocytosis. Therefore, balanced levels of EHD1-EHD4 are important for NgCAM endocytosis in neurons. Our data suggest that EHD1 plays roles in both endosomal recycling and a specialized endocytosis pathway in neurons used by NgCAM. We propose that EHD1 and EHD4 act as hetero-oligomeric complexes in this pathway.
Project description:Endocytosis defines the entry of molecules or macromolecules through the plasma membrane as well as membrane trafficking in the cell. It depends on a large number of proteins that undergo protein-protein and protein-phospholipid interactions. EH Domain containing (EHDs) proteins formulate a family, whose members participate in different stages of endocytosis. Of the four mammalian EHDs (EHD1-EHD4) EHD1 and EHD3 control traffic to the endocytic recycling compartment (ERC) and from the ERC to the plasma membrane, while EHD2 modulates internalization. Recently, we have shown that EHD2 undergoes SUMOylation, which facilitates its exit from the nucleus, where it serves as a co-repressor. In the present study, we tested whether EHD3 undergoes SUMOylation and what is its role in endocytic recycling. We show, both in-vitro and in cell culture, that EHD3 undergoes SUMOylation. Localization of EHD3 to the tubular structures of the ERC depends on its SUMOylation on lysines 315 and 511. Absence of SUMOylation of EHD3 has no effect on its dimerization, an important factor in membrane localization of EHD3, but has a dominant negative effect on its appearance in tubular ERC structures. Non-SUMOylated EHD3 delays transferrin recycling from the ERC to the cell surface. Our findings indicate that SUMOylation of EHD3 is involved in tubulation of the ERC membranes, which is important for efficient recycling.
Project description:EHD proteins have been implicated in intracellular trafficking, especially endocytic recycling, where they mediate receptor and lipid recycling back to the plasma membrane. Additionally, EHDs help regulate cytoskeletal reorganization and induce tubule formation. It was previously shown that EHD proteins bind directly to the C2 domains in myoferlin, a protein that regulates myoblast fusion. Loss of myoferlin impairs normal myoblast fusion leading to smaller muscles in vivo but the intracellular pathways perturbed by loss of myoferlin function are not well known. We now characterized muscle development in EHD1-null mice. EHD1-null myoblasts display defective receptor recycling and mislocalization of key muscle proteins, including caveolin-3 and Fer1L5, a related ferlin protein homologous to myoferlin. Additionally, EHD1-null myoblast fusion is reduced. We found that loss of EHD1 leads to smaller muscles and myofibers in vivo. In wildtype skeletal muscle EHD1 localizes to the transverse tubule (T-tubule), and loss of EHD1 results in overgrowth of T-tubules with excess vesicle accumulation in skeletal muscle. We provide evidence that tubule formation in myoblasts relies on a functional EHD1 ATPase domain. Moreover, we extended our studies to show EHD1 regulates BIN1 induced tubule formation. These data, taken together and with the known interaction between EHD and ferlin proteins, suggests that the EHD proteins coordinate growth and development likely through mediating vesicle recycling and the ability to reorganize the cytoskeleton.
Project description:The four highly homologous human EHD proteins (EHD1-4) form a distinct subfamily of the Eps15 homology domain-containing protein family and are thought to regulate endocytic recycling. Certain members of this family have been studied in different cellular contexts; however, a lack of concurrent analyses of all four proteins has impeded an appreciation of their redundant versus distinct functions.Here, we analyzed the four EHD proteins both in mammalian cells and in a cross-species complementation assay using a C. elegans mutant lacking the EHD ortholog RME-1. We show that all human EHD proteins rescue the vacuolated intestinal phenotype of C. elegans rme-1 mutant, are simultaneously expressed in a panel of mammalian cell lines and tissues tested, and variably homo- and hetero-oligomerize and colocalize with each other and Rab11, a recycling endosome marker. Small interfering RNA (siRNA) knock-down of EHD1, 2 and 4, and expression of dominant-negative EH domain deletion mutants showed that loss of EHD1 and 3 (and to a lesser extent EHD4) but not EHD2 function retarded transferrin exit from the endocytic recycling compartment. EH domain deletion mutants of EHD1 and 3 but not 2 or 4, induced a striking perinuclear clustering of co-transfected Rab11. Knock-down analyses indicated that EHD1 and 2 regulate the exit of cargo from the recycling endosome while EHD4, similar to that reported for EHD3 (Naslavsky et al. (2006) Mol. Biol. Cell 17, 163), regulates transport from the early endosome to the recycling endosome.Altogether, our studies suggest that concurrently expressed human EHD proteins perform shared as well as discrete functions in the endocytic recycling pathway and lay a foundation for future studies to identify and characterize the molecular pathways involved.