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

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.羧基端Eps15同源结构域蛋白（EHD1-4）在调控胞吞运输过程中发挥关键作用。EHD2是该家族中唯一已解析晶体结构的成员，其包含一段由两个脯氨酸-苯丙氨酸（PF）基序组成的无结构环：KPFRKLNPF。与之相反，尽管EHD2与其旁系同源蛋白EHD1、EHD3和EHD4的氨基酸序列同一性高达近70%，但后三者仅含有单个KPF或RPF基序，并不含NPF基序。本研究旨在明确EHD2各PF基序在其同源二聚化、蛋白伴侣结合以及亚细胞定位中的具体作用。为验证NPF基序的功能，我们构建了EHD2 NPF突变为NAF的突变体，以模拟EHD1和EHD3的同源序列。我们证实该突变体丧失了同源二聚化以及结合Syndapin2的能力，但仍主要定位于质膜的胞浆侧。与之相对，EHD2 NPF突变为APA的突变体则保留了正常的同源二聚化与Syndapin2结合能力，但其核定位显著增强，且与质膜的结合减少。随后我们提出假说：EHD1中与EHD2的KPF基序相对应的单个PF基序，可能负责EHD1的结合与定位功能。实验证实，EHD1的RPF基序是其同源二聚化、与MICAL-L1和Syndapin2相互作用以及定位于管状循环内体所必需的。此外，循环实验结果显示，EHD1 RPF突变为APA的突变体无法支持正常的受体循环过程。综上，我们的实验数据表明，EHD2 NPF基序中的苯丙氨酸残基对于EHD2定位于质膜至关重要，而脯氨酸残基则是其同源二聚化与蛋白结合所必需的。本研究支持近期提出的模型：EHD2的N端区域可调控无结构环与邻近EHD2二聚体相互作用的可及性，从而促进寡聚化过程。