Molecular mechanisms of C-terminal Eps15 Homology Domain containing (EHD) protein function

Endocytic trafficking is not only an essential process for the maintenance of cellular homeostasis but also plays a vital role in regulating diverse cellular processes such as signaling, migration and cell division. The C-terminal Eps 15 Homology Domain proteins (EHD1-4) play pivotal roles in regulating distinct steps of endocytic trafficking. Among the EHDs, EHD2 is disparate both in terms of sequence homology (70%) and its subcellular localization at the caveolae. The crystal structure of EHD2 has been solved and it contains an unstructured loop consisting of two proline-phenylalanine (PF) motifs: KPFRKLNPF. However, the other paralogs EHD1, EHD3 and EHD4 contain a single KPF or RPF motif, but no NPF motif. In this study, we sought to elucidate the precise role of the two PF motifs of EHD2 in homo-dimerization, binding with the protein partners, and subcellular localization. We demonstrated that an EHD2 NPF-to-NAF mutant that mimics the homologous sequences of EHD1 and EHD3, lost its ability to dimerize and bind to Syndapin2. However, it continues to localize primarily to the cytosolic face of the plasma membrane. On the other hand, EHD2 NPF-to-APA mutants maintained their ability to dimerize and bind to Syndapin2, but exhibited markedly increased nuclear localization and decreased association with the plasma membrane. Hence, 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 also 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. We further hypothesized that the single PF motif of EHD1 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 on tubular recycling endosomes. Moreover, recycling assays demonstrated that EHD1 RPF-to-APA was incapable of supporting normal receptor recycling. The biogenesis of tubular recycling endosomes (TRE), their role in cargo-sorting and subsequently their vesiculation are essential for receptor recycling. EHD proteins have been implicated in the bending and fission of TRE, thus regulating endocytic recycling. Recent studies from our lab have demonstrated that asparagine-proline-phenyalanine (NPF)-containing binding partners of EHD1 and EHD3, such as molecules interacting with CasL-like1 (MICAL-L1) and Syndapin2, are indispensable for TRE biogenesis. Also vital for TRE biogenesis is the generation of phosphatidic acid (PA), an essential lipid component of TRE that serves as a docking point for MICAL-L1 and Syndapin2. EHD1 and EHD3 have 86% amino acid identity; they homo-and heterodimerize and partially co-localize to TRE. Despite remarkable identity between EHD1 and EHD3, they have disparate mechanistic functions. EHD1 induces membrane vesiculation, whereas EHD3 supports TRE generation and/or stabilization by an unknown mechanism. While using phospholipase D inhibitors (which block the conversion of glycerophospholipids to PA) to deplete cellular TRE, we observed that, upon inhibitor washout, there was a rapid and dramatic regeneration of TRE, as observed by immunostaining with MICAL-L1 antibodies. This “synchronized” TRE biogenesis system has enabled us to determine that EHD3 is involved in the stabilization of TRE rather than in their biogenesis. Moreover, we have identified residues Ala-519/Asp-520 in the EH domain of EHD1 and Asn-519/ Glu-520 in the EH domain of EHD3 as being important for that dictating the preference of these two paralogs for NPF-containing binding partners. Overall, we have delineated a model to explain the atomic basis for understanding the differential roles of EHD3 and EHD1 in stabilization and vesiculation of TRE, respectively

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

<div><p>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: <b>KPF</b>RKL<b>NPF</b>. 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.</p></div

A single EHD1 PF motif (RPF) controls its homo- and hetero-dimerization, binding to interaction partners, and localization to Tubular Recycling Endosomes (TRE).

<p>(A) <i>S</i>. <i>cerevisae</i> yeast were co-transformed with Gal4bd fusion constructs: Gal4bd-p53 (control), -Syndapin-2 and -MICAL-L1 along with Gal4ad-SV40 (control), -EHD1 (wt.) and -EHD1 (RPF-to-APA). (B) As in (A), yeast were co-transformed with Gal4bd-p53 (control), -EHD1 (wt), -EHD3 (wt), -EHD4 (wt), along with Gal4ad-SV40 (control), -EHD1 (wt) and EHD1 (RPF-to-APA). Co-transformants from A-B were plated on non-selective (+HIS) and selective (-HIS) agar plates. (C–D) HeLa cells were grown on coverslips, transfected with GFP-myc-EHD1 (wt) (C), GFP-myc-EHD1 (RPF-to-APA) (D, arrows point to TRE). Bar; 10 μm.</p

The NAF motif of EHD1 is dispensable for homo- or hetero-oligomerization, and for its association with binding partners.

<p>(A) <i>S</i>. <i>cerevisae</i> yeast were co-transformed with the following Gal4bd fusion constructs: Gal4bd-p53 (control), -MICAL-L1, and -Syndapin-2 along with Gal4ad-SV40 (control), -EHD1 (wt.) and -EHD1 (NAF to NPF). (B) As in (A), yeast were co-transformed with Gal4bd-p53 (control), -EHD1 (wt), -EHD2 (wt), -EHD3 (wt) and -EHD4 (wt), along with Gal4ad-SV40 (control), -EHD1 (wt) and -EHD1 (NAF-to-NPF). (C) As in (A), yeast were co-transformed with Gal4bd-p53 (control), -EHD1 (wt), -EHD3 (wt), and -EHD4 (wt), along with Gal4ad-SV40 (control), -EHD1 (wt) and -EHD1 (NAF-to-NPF). (D) Yeast were co-transformed with Gal4bd-p53 (control), -Syndapin-2, and -MICAL-L1, along with Gal4ad-SV40 (control), EHD1 (wt) and EHD1 (NAF-to-NPF). Co-transformants in A-D were plated on non-selective (+HIS) and selective (-HIS) agar plates.</p

EHD protein domain architecture and sequence homology.

<p>(A) The Eps15 Homology Domain (EHD) proteins have a conserved domain architecture comprised of four domains: two helical domains, a G-domain, and a C-terminal EH domain. The G-domain of EHD2 contains an unstructured loop containing the following amino acid sequence, KPFRKLNPF, which is required for oligomerization. (B) The four EHD isoforms share 67–86% residue identity. The amino acid sequence alignment of the unstructured loop for all four EHDs (see green frame) shows that only EHD2 has two successive <b>PF</b> motifs: N<b>PF</b> and K<b>PF</b>, whereas the other EHD proteins have only one <b>PF</b> motif.</p

Modification of the EHD2 NPF motif to APA induces loss of plasma membrane localization, but does not affect interactions with binding partners.

<p>(A) <i>S</i>. <i>cerevisae</i> yeast were co-transformed with the following Gal4bd fusion constructs: Gal4bd-p53 (control), -Syndapin-2 and -MICAL-L1 along with Gal4ad fusion constructs Gal4ad-SV40 (control), -EHD2 (wt.) and -EHD2 (NPF-to-APA). (B) As in (A) co-transformation was with Gal4bd fusion constructs: Gal4bd-p53 (control), -EHD2 (wt) along with Gal4ad fusion constructs: Gal4ad-SV40 (control), -EHD2 (wt) and -EHD2 (NPF-to-APA). Co-transformants from A-B were plated on non-selective (+HIS) and selective (-HIS) agar plates. (C–D) HeLa cells were grown on coverslips, transfected with GFP-myc-EHD2 (C) or GFP-myc-EHD2 NPF-to-APA (D). Bar; 10 μm.</p

The NPF phenylalanine residue is responsible for the plasma membrane localization of EHD2.

<p>(A) <i>S</i>. <i>cerevisae</i> yeast were co-transformed with the following Gal4bd fusion constructs: Gal4bd-p53 (control), -Syndapin-2 and -MICAL-L1 along with Gal4ad fusion constructs: Gal4ad-SV40 (control), -EHD2 (wt) and -EHD2 (NPF-to-NPA). (B) As in (A), yeast were co-transformed with the Gal4bd fusion constructs: Gal4bd-p53 (control), -EHD2 (wt) along with Gal4ad fusion constructs: Gal4ad-SV40 (control), -EHD2 (wt.) and -EHD2 (NPF-to-NPA). Co-transformants were plated on non-selective (+HIS) and selective (-HIS) agar plates. (C–I) HeLa cells were grown on coverslips and transfected with: (C) GFP-myc-EHD2 (wt), (D) GFP-myc-EHD2 (NPF-to-NPA), (E) GFP-myc-EHD2 (NPF-to-NPY), (F) GFP-myc-EHD2 (NPF-to-NPA), (G) GFP-myc-EHD2 (NPF-to-NFP). Bar; 10 μm.</p

Disruption of the EHD2 KPF motif induces relocalization of EHD2 to the nucleus, but does not alter its binding ability.

<p>(A–D) <i>S</i>. <i>cerevisae</i> yeast were co-transformed with the following Gal4bd fusion constructs: Gal4bd-p53 (control), -Syndapin-2 and -MICAL-L1 along with Gal4ad fusion constructs: Gal4ad-SV40 (control), -EHD2 (wt), -EHD2 (KPF-to-APA) and—EHD2 (KPF-to-KAF). Co-transformants were plated on non-selective (+HIS) and selective (-HIS) media. (E–H) HeLa cells were grown on coverslips, transfected with GFP-myc-EHD2 (wt) (E), GFP-myc-EHD2 (KPF-to-APA) (F), or GFP-myc-EHD2 (KPF-to-KAF) (G) and analyzed by confocal microscopy. Bar; 10 μm.</p