The neuromuscular junction (NMJ) is a subcellular specialization of the myofiber plasma membrane, with nuclear domains directing synaptic gene expression. Our long-term goal is to provide a more complete molecular model of the NMJ. We previously identified concordance of the mammalian NMJ protein components with those of the Torpedo californica electric organ, describing the developmental origins of the organ and its extreme development into an amplified cholinergic synapse relative to skeletal muscle, to support its use as a model NMJ for hypothesis generation . We identified several high-abundance proteins including Eps 15 homology domain-containing 1 (EHD1; Swiss-Prot:Q5E9R3), adducin gamma (ADD3; Swiss-Prot:Q9UEY8), laminin receptor protein 1 (LamR1; Swiss-Prot:Q803F6), chromosome 1 open reading frame 123 (C1orf123; Swiss-Prot: Q9NWV4), transgelin-3 (TAGL3; Swiss-Prot: P37805), and transforming growth factor-β-induced (TGFBI; Swiss-Prot:Q15582), which may play a role in synapse structure and maintenance. This approach of using the proteomic profile of an amplified model synapse should expedite candidate NMJ protein identification and characterization and thus help inconstructing a more complete NMJ paradigm.
In the current study, EHD1 was examined because of the high number of unique peptides (n = 20) identified in the electric organ proteome relative to mouse skeletal muscle (n = 0), and its high spectral cross-correlation value (140). In addition, EHD1 was investigated as a peripheral membrane protein that functions in clathrin-independent endocytosis and recycling of receptors at the membrane through the tubular endosomal recycling compartment (ERC) [1, 2].
The EHD family of proteins (EHD1 to EHD4) contain an EH domain that facilitates interactions with proteins encoding asparagine-proline-phenylalanine (NPF) motifs, which form complexes that regulate endocytic trafficking [3, 4]. The current functional paradigm for this group of proteins is that EHD3 and EHD4 assist in the transport of proteins from the early endosome (EE) into the ERC whereas EHD1 and EHD2 assist in the cargo exit from the ERC to the plasma membrane . In addition to the C-terminal EH domain that EHD proteins share with many proteins of the endocytic machinery, EHD family proteins share a central coiled-coil and an N-terminal phosphate binding loop (P-loop) [3, 5]. These proteins are products of gene duplication, are encoded on separate chromosomes, and have differential expression profiles in various tissues [3, 4, 6–8]. In adult tissues, EHD1 is expressed in germ cells, adipocytes, the eye (retina, rods and cones outer nuclear layer, internal nuclear layer, and ganglion cell layer), the basal membrane of the endometrium and uterine muscle cells, granulosa cells after ovulation, skeletal muscle, kidney, and spermatocytes, but it has not been found in spleen, liver, or brain . The EHD1 protein has been studied in multiple cultured cells, whole-tissue extracts, and the testis; however, its subcellular localization in normal tissues has not been characterized.
Several proteins known to serve as components of presynaptic and postsynaptic membranes contain NPF domains, suggesting their potential interaction with the EH domain of EHD1 and/or other family members. At the presynaptic membrane these include stoned (stnB), synaptosomal-associated protein (Snap)29, secretory carrier membrane proteins (SCAMP)1 and SCAMP5, and syndapin I (also known as Pacsin I). Each of these proteins functions as part of the syanaptosome that regulates vesicle transport and neurotransmitter release across the NMJ [9–14]. Interestingly, the EH domain of EHD1 binds snapin, a soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE)-associated protein that does not contain an NPF motif, effectively blocking exocytosis of synaptic vesicle . Furthermore, neuronal-glial cell adhesion molecule (NgCAM) trafficking is dependent on EHD1 . Overall, EHD proteins are thought to be an important component of the presynaptic synaptosome.
In the postsynaptic membrane, ankyrins are known to stabilize membrane and membrane-associated proteins at the NMJ . Expression of EHD1-4 proteins was increased in ankyrin-B−/− cardiomyocytes . Functional studies in HeLa cells showed that EHD1 regulates the expression of β1 integrin via a clathrin-independent mechanism and Arf6 and Rab family proteins . β1 integrin is a key extracellular-matrix (ECM) receptor that facilitates interactions with the ECM at focal adhesions but is also a key mediator of downstream signaling pathways important for cell survival, growth, environmental sensing, and cellular movement [19, 20]. Furthermore, β1 integrin has high expression in skeletal muscle and forms a dimer with α7 integrin at the NMJ, where it synergistically interacts with agrin and laminins 1 and 2/4 to promote acetylcholine receptor (AChR) clustering during maturation of muscle and AChR stability throughout life , supporting the potential role of EHD1 at the NMJ.
Maintenance and signaling of postsynaptic receptors is intimately linked to their turnover and trafficking, suggesting a possible role for EHD1 and its paralogs. Both ErbB receptor tyrosine kinases and muscle-specific tyrosine-protein kinase receptors (MuSK) are thought to signal after endocytosis into vesicles containing the downstream proteins that initiate synaptic gene transcription, reorganization of the cytoskeletal network, and clustering of AChRs or gene transcription (the so-called signaling endosome hypothesis). Earlier studies showed that ligand-induced endocytosis of MuSK occurs via a clathrin-independent but lipid-raft-dependent pathway [22, 23]. By contrast, ErbB receptor tyrosine kinases are internalized through the clathrin-mediated endocytic pathway upon neuregulin binding to activate AChR expression . Interestingly, the NPF-domain containing protein phosphatidylinositol-binding clathrin assembly protein (CALM) functions in AP-2-dependent clathrin-mediated endocytosis , potentially by intersecting with EHD proteins. Furthermore, tyrosine phosphorylation is crucial for recycling of endocytosed AChR to the synaptic crests; inhibitors of tyrosine phosphorylation cause AChR to become trapped or located in perisynaptic regions .
Given the role of EHD1 in the transport of ligand-bound receptors, and given the importance of ligand-bound receptors such as MuSK and ErbB in postsynaptic stabilization and gene expression, we hypothesized that EHD proteins might play an important role in AChR clustering and postsynaptic membrane architecture. We report the localization and function of a previously unknown NMJ protein, EHD1.