Alternative titles; symbolsEPSINHGNC Approved Gene Symbol: EPN1Cytogenetic location: 19q13.42 Genomic coordinates (GRCh38): 19:55,675,225-55,709,532 (from NC...
Alternative titles; symbols
HGNC Approved Gene Symbol: EPN1
Cytogenetic location: 19q13.42 Genomic coordinates (GRCh38): 19:55,675,225-55,709,532 (from NCBI)
EPN1 is an endocytic accessory protein that interacts with EPS15 (600051), the alpha subunit of the clathrin adaptor AP2 (AP2A1; 601026), and clathrin (see 118960), as well as with other accessory proteins for the endocytosis of clathrin-coated vesicles.
▼ Cloning and Expression
Chen et al. (1998) cloned rat Epn1 from a rat brain cDNA library. The deduced 576-amino acid protein contains several conserved asp-pro-trp (DPW) repeats in its central region, a Cdc2 (116940) phosphorylation site, and 3 arg-pro-phe (NPF) repeats in its C-terminal region. Northern blot analysis detected a 2.6-kb transcript in all rat tissues tested, and Western blot analysis confirmed ubiquitous expression. Immunofluorescence localization using rat brain frozen sections revealed colocalization of Epn1 with Eps15, synaptophysin (313475), and clathrin in nerve terminals.
Morinaka et al. (1999) identified EPN1 as an 84-kD protein that binds bovine brain Pob1 (REPS2; 300317). By searching an EST database, PCR, and 5-prime and 3-prime RACE of a brain cDNA library, they cloned human EPN1. The deduced 551-amino acid protein contains an epsin N-terminal homology (ENTH) region, which includes 1 DPW sequence, followed by 8 central DPW repeats, a single LVDLD sequence, and 3 NPF repeats at the C terminus. The LVDLD sequence is a clathrin-binding motif, and NPF is the core motif of an EPS15 homology (EH) domain-binding domain.
▼ Gene Function
Using truncation mutants of rat brain Epn1, Chen et al. (1998) determined that the central region of Epn1, which contains the DPW repeats, binds AP2, and the C-terminal region binds Eps15. Rat Epn1 associated with clathrin coats in situ and could be coprecipitated with AP2 and Eps15. It did not copurify with mature clathrin-coated vesicles. Chen et al. (1998) concluded that EPN1 may participate with EPS15 in the molecular rearrangement of the clathrin coats that are required for coated-pit invagination and vesicle fission.
Using in vitro pull-down assays, Morinaka et al. (1999) determined that the EH domain of REPS2 interacts specifically with a 10-amino acid C-terminal peptide of EPN1 containing the NPF sequence. Expression of EPN1 in CHO cells overexpressing insulin receptor (INSR; 147670) inhibited internalization of insulin, but it did not affect insulin-binding or autophosphorylation of INSR.
Kariya et al. (2000) found that EPN1, RALA-binding protein-1 (RALBP1; 605801), REPS2, and EPS15 formed a complex with AP2A1 in CHO cells. They noted that EPN1 contains a single putative phosphorylation site at ser357, and they transfected human EPN1 containing a point mutation (ser357 to asp) at this site into CHO cells overexpressing INSR. Phosphorylated EPN1 and the ser357-to-asp mutant formed a complex with AP2A1 less efficiently than nonphosphorylated wildtype EPN1. Phosphorylation of EPN1 also inhibited binding with the EH domain of REPS2, suggesting that phosphorylation of EPN1 inhibits receptor-mediated endocytosis by disassembly of its complex with REPS2 and AP2A1.
Ford et al. (2002) found that EPN1 expressed in transfected COS cells showed a general cytoplasmic distribution or accumulation in puncta on the plasma membrane. EPN1 colocalized with AP2, clathrin, EPS15, and dynamin (see 602377). The presence of dynamin suggested that the puncta represent endocytically incompetent coated pits. Ford et al. (2002) determined that the ENTH domain of EPN1 binds the membrane lipid phosphatidylinositol-4,5-bisphosphate, or PtdIns(4,5)P2. Monomeric EPN1 modified membrane curvature upon binding to PtdIns(4,5)P2. On lipid monolayers, EPN1 alone was sufficient to facilitate the formation of clathrin-coated invaginations.
Using fluorescence microscopy, Chen and Zhuang (2008) identified EPN1 as a cargo-specific adaptor for influenza virus entry through the clathrin-mediated pathway. EPN1 was recruited to virus-binding sites in synchrony with the assembly of clathrin-coated pits. Knockdown of EPN1 by small interfering RNA inhibited clathrin-mediated endocytosis and caused the virus to enter through a clathrin-independent pathway. EPN1 was not required for clathrin-mediated endocytosis of transferrin (TF; 190000), EGF, and low density lipoprotein.
▼ Biochemical Features
Endocytic proteins such as epsin, AP180 (603025), and HIP1R (605613) share a conserved modular region termed the ENTH domain, which plays a crucial role in clathrin-mediated endocytosis. Itoh et al. (2001) demonstrated a strong affinity of the ENTH domain for PtdIns(4,5)P2. With nuclear magnetic resonance analysis of the epsin ENTH domain, they determined that a cleft formed with positively charged residues contributed to phosphoinositide binding. Overexpression of a mutant, epsin lys76 to ala, with an ENTH domain defective in phosphoinositide binding blocked epidermal growth factor internalization in COS-7 cells. Thus, interaction between the ENTH domain and PtdIns(4,5)P2 is essential for endocytosis mediated by clathrin-coated pits.