STRUCTURAL ANALYSIS OF EHD1

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 C-末端Eps15同源结构域蛋白EHD1调节受体从内吞循环到质膜的循环。在细胞中，EHD1定位于管状和球状再循环内小体。到目前为止，EHD1与内膜结合的方式尚不清楚，也没有确定这种相互作用是直接的还是通过相互作用的蛋白质。在这里，我们提供的证据表明，EHD1具有直接和优先结合一系列磷脂的能力，首选磷脂酰肌醇脂类，在第3位有磷酸。先前的研究表明，EH结构域与钙结合并与含有天冬氨酸-脯氨酸-苯丙氨酸(NPF)三肽的蛋白质相互作用。利用二维核磁共振分析，我们现在描述了EHD1的Eps15同源(EH)结构域的一个新功能，并表明它能够直接结合磷脂酰肌醇部分。此外，我们扩大了我们的研究范围，包括EHD4的C端EH结构域和Eps15的三个N端EH结构域中的第二个，并证明磷脂酰肌醇结合可能是其他EH结构域所共有的更一般的特性。我们的分析揭示了一个模型，通过这个模型，在一个30个氨基酸的延伸中，几个带正电的残基的侧链主要定位在EH-结构域的第二和第三螺旋上，介导与磷脂酰肌醇的直接相互作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The C-terminal Eps15 homology domain-containing protein, EHD1, regulates the recycling of receptors from the endocytic recycling compartment to the plasma membrane. In cells, EHD1 localizes to tubular and spherical recycling endosomes. To date, the mode by which EHD1 associates with endosomal membranes remains unknown, and it has not been determined whether this interaction is direct or via interacting proteins. Here, we provide evidence demonstrating that EHD1 has the ability to bind directly and preferentially to an array of phospholipids, preferring phosphatidylinositol lipids with a phosphate at position 3. Previous studies have demonstrated that EH-domains coordinate calcium binding and interact with proteins containing the tripeptide asparagine-proline-phenylalanine (NPF). Using two-dimensional Nuclear Magnetic Resonance analysis, we now describe a new function for the Eps15 homology (EH) domain of EHD1 and show that it is capable of directly binding phosphatidylinositol moieties. Moreover, we have expanded our studies to include the C-terminal EH-domain of EHD4 and the second of the three N-terminal EH-domains of Eps15, and have demonstrated that phosphatidylinositol-binding may be a more general property shared by other EH-domains. Our analysis reveals a model by which the side chains of several positively charged residues within a 30 amino acid stretch localized primarily to the second and third helices of the EH-domain mediate a direct interaction with phosphatidylinositols.