Role of the EHD2 Unstructured Loop in Dimerization, Protein Binding and Subcellular Localization

Abstract

The C-terminal Eps 15 Homology Domain proteins (EHD1-4) play important roles in regulating endocytic trafficking. EHD2 is the only family member whose crystal structure has been solved, and it contains an unstructured loop consisting of two proline-phenylalanine (PF) motifs: KPFRKLNPF. In contrast, despite EHD2 having nearly 70% amino acid identity with its paralogs, EHD1, EHD3 and EHD4, the latter proteins contain a single KPF or RPF motif, but no NPF motif. In this study, we sought to define the precise role of each PF motif in EHD2’s homo-dimerization, binding with the protein partners, and subcellular localization. To test the role of the NPF motif, we generated an EHD2 NPF-to-NAF mutant to mimic the homologous sequences of EHD1 and EHD3. We demonstrated that this mutant lost both its ability to dimerize and bind to Syndapin2. However, it continued to localize primarily to the cytosolic face of the plasma membrane. On the other hand, EHD2 NPF-to-APA mutants displayed normal dimerization and Syndapin2 binding, but exhibited markedly increased nuclear localization and reduced association with the plasma membrane. We then hypothesized that the single PF motif of EHD1 (that aligns with the KPF of EHD2) might be responsible for both binding and localization functions of EHD1. Indeed, the EHD1 RPF motif was required for dimerization, interaction with MICAL-L1 and Syndapin2, as well as localization to tubular recycling endosomes. Moreover, recycling assays demonstrated that EHD1 RPF-to-APA was incapable of supporting normal receptor recycling. Overall, our data suggest that the EHD2 NPF phenylalanine residue is crucial for EHD2 localization to the plasma membrane, whereas the proline residue is essential for EHD2 dimerization and binding. These studies support the recently proposed model in which the EHD2 N-terminal region may regulate the availability of the unstructured loop for interactions with neighboring EHD2 dimers, thus promoting oligomerization.

Dictyostelium EHD associates with Dynamin and participates in phagosome maturation

“…D. discoideum EHD contains only the PF motif in this loop (Fig. 1A), like human EHD1 in which this motif is sufficient for oligomerization (Bahl et al, 2015). In mouse EHD2, an additional GPF motif has been identified in the linker region between the G domain and the EH domain and is predicted to bind to the EH domain of the opposing dimeric EHD2 molecule (Daumke et al, 2007).…”

“…Cells were fixed for 1 h in 2% glutaraldehyde in HL5c culture medium and then stained for 30 min in 2% osmium with 0.1 M imidazole, as described previously (Barisch et al, 2015). After staining, cells were washed with PBS.…”

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

“…Moreover, EHD4 hetero-dimerized with EHD3 (which displays 86% identity with EHD1) and this interaction was similarly abrogated by the EHD3 V203P coil-breaking mutant (Fig 1D). However, EHD4 was unable to hetero-dimerize with EHD2 (which displays only 67% identity with EHD1 and diverges significantly from the functions of its other paralogs [50][51][52][53][54][55][56][57][58][59][60]) ( Fig 1D).…”

Prognostic implication of histological features associated with EHD2 expression in papillary thyroid carcinoma

“…Endocytosis is required to the transport nutrients into a cell, cell adhesion, cell migration, and receptor-mediated signaling [ 14 – 16 ]. C-terminal Eps 15 homology domain (EHD) proteins are involved in the regulation of endocytic membrane trafficking [ 14 – 16 ].…”

“…Endocytosis is required to the transport nutrients into a cell, cell adhesion, cell migration, and receptor-mediated signaling [ 14 – 16 ]. C-terminal Eps 15 homology domain (EHD) proteins are involved in the regulation of endocytic membrane trafficking [ 14 – 16 ]. Of four mammalian EHD proteins (EHD1-4), EHD2 participates in clathrin-dependent endocytosis and endosomal recycling by regulating caveolar mobility and controlling Rac1 and actin cytoskeleton [ 16 ].…”