Regulation of EHD protein function by molecular partner interactions

描述(由申请人提供)：控制受体定位到质膜是正常细胞功能的核心，调节失调是导致动脉粥样硬化、糖尿病和癌症等各种疾病的根本原因。质膜上的受体水平取决于内化和再循环回到细胞表面的速率。我们和其他人最近发现了一种新的含有Eps15同源(EH)结构域的蛋白质，EHD1，它是内吞循环途径的关键组成部分。EHD介导的功能在生理上的重要性已被清楚地证明，因为EHD1功能的丧失会导致许多关键受体的循环受损。该项目的长期目标是了解控制蛋白质在细胞内运输和向质膜运输的基本机制，重点是了解EHD结构影响其功能的方式。这一提议的中心假设是，EHD1(EH-1)的EH结构域及其C端EHD类似物的静电表面电荷负责与含有NPF的蛋白质伙伴和磷脂酰肌醇相互作用的选择性，并且这些相互作用对于调节细胞内转运是重要的。我们基于强大的初步数据为我们的假设提供了令人信服的证据，即EH-1选择性地与含有NPF基序的蛋白质相互作用，随后是一簇带负电荷的残基。此外，我们的新的初步数据也反映了EHD/磷脂酰肌醇相互作用的功能重要性，证明了EH-1能够直接与磷脂酰肌醇部分相互作用，并且这种相互作用是允许EHD1定位于管状和囊泡膜结构所必需的。我们提出这一建议的具体目的是：1)确定特定含NPF蛋白的C-末端EH结构域选择性的分子和结构基础；2)阐明富含/组成EHD管膜的特定磷脂酰肌醇(S)，并确定EHD/磷脂酰肌醇结合在调节细胞内转运中的意义。从这项研究中获得的知识将为C-末端EHD蛋白与内体膜结合的方式和协调循环的控制提供关键的新信息。这将大大提高对调节受体循环的内吞机制的理解，并将对健康和疾病产生重要影响。

DESCRIPTION (provided by applicant): Control of receptor localization to the plasma membrane is central to normal cell function, and dysregulation is the underlying cause for diseases as diverse as atherosclerosis, diabetes and cancer. Receptor levels on the plasma membrane are dependent upon the rates of internalization and recycling back to the cell surface. We and others have recently identified a novel Eps15 homology (EH)-domain containing protein, EHD1, as a critical component of the endocytic recycling pathway. The physiologic importance of EHD-mediated function has been clearly demonstrated, as loss of EHD1 function leads to impaired recycling of many critical receptors. The long- term goals of this project are to understand the fundamental mechanisms controlling intracellular trafficking and transport of proteins to the plasma membrane, with emphasis on understanding the mode by which EHD structure impacts its function. The central hypothesis of this proposal is that the electrostatic surface charge of the EH-domain of EHD1 (EH-1) and its C-terminal EHD paralogs is responsible for the selectivity of interactions with NPF-containing protein partners and phosphoinositides, and that these interactions are important for the regulation of endocytic transport. We provide compelling evidence for our hypothesis based on our strong preliminary data that EH-1 selectively interacts with proteins containing NPF motifs followed by a cluster of negatively charged residues. In addition, our new preliminary data also reflect the functional importance of the EHD/phosphoinositide interactions by demonstrating that EH-1 is capable of interacting directly with phosphatidylinositol moieties, and that this interaction is required to allow localization of EHD1 to tubular and vesicular membrane structures. Our Specific Aims for the proposal are: 1) To identify the molecular and structural basis for the selectivity of C-terminal EH-domains for specific NPF-containing proteins, and 2) To elucidate the specific phosphoinositide(s) that are enriched/comprise EHD tubular membranes and determine the significance of EHD/phosphoinositide binding in the regulation of endocytic trafficking. The knowledge to be obtained from this study will provide critical new information on the mode by which the C-terminal EHD proteins associate with endosomal membranes and coordinate control of recycling. This will lead to a significantly enhanced understanding of the endocytic mechanisms that regulate receptor recycling, and will have an important bearing on health and disease.