Molecular mechanisms controlling endocytic recycling

描述（由申请人提供）：受体内化和再循环至质膜的控制是正常细胞功能的核心，这些过程的失调是动脉粥样硬化、糖尿病和癌症等多种疾病的根本原因。在关键的内吞调节蛋白中有超过50种Rab-GTP结合蛋白及其控制囊泡运输和融合事件的效应物。在过去的五年里，我们一直专注于了解一种新的内吞调节蛋白家族的作用，称为C-末端Eps 15同源结构域蛋白（EHD）。尽管越来越多的研究表明EHD蛋白在调节多种受体的转运中的作用，但迄今为止，EHD蛋白的实际机制功能的统一假设仍然难以捉摸。EH-结构域与含有三肽基序天冬酰胺-脯氨酸-苯丙氨酸（NPF）的蛋白质相互作用。最重要的是，我们发现了一种称为与CasL-Like 1相互作用的分子（MICAL-L1）的蛋白质是EHD 1的一种新型含NPF的相互作用伴侣。MICAL-L1作为不寻常的Rab效应物起作用，因为它对于Rab 8a和EHD 1向富含磷脂酰肌醇-4-磷酸和磷脂酰肌醇-4，5-二磷酸的肾小管再循环内体的募集是关键的。MICAL-L1的消耗模拟EHD 1消耗，导致再循环至质膜的延迟和内化受体在内吞再循环室（ERC）处的积累。此外，新的初步数据表明，MICAL-L1还与膜弯曲BAR结构域蛋白Syndapin II相互作用。总的来说，我们的数据表明MICAL-L1在调节管状内体的产生和招募EHD 1中起关键作用，EHD 1似乎负责其随后的囊泡形成。我们的中心假设是EHD 1在肾小管膜断裂中起着关键作用，并促进膜和蛋白质向质膜的再循环。我们的第一个具体目标是确定EHD 1在小管膜断裂和再循环中发挥作用的机制。我们的工作假设是，ATP水解EHD 1促进切割囊泡从EHD 1含有小管，从而支持回收到质膜。我们的第二个具体目标是描述选择的EHD 1相互作用伴侣在小管膜生成和调节EHD 1功能中的作用。我们假设，BAR结构域的EHD 1相互作用伙伴（如Syndapin II和/或Bin 1）产生的EHD 1被招募到管状膜。我们进一步假设MICAL-L1通过维持其在肾小管膜上而在调节EHD 1功能中起着至关重要的作用。最终，这些研究将促进新策略的发展，以治疗由于异常内吞事件而引起的许多疾病。

DESCRIPTION (provided by applicant): Control of receptor internalization and recycling to the plasma membrane is central to normal cell function, and dysregulation of these processes is the underlying cause for diseases as diverse as atherosclerosis, diabetes, and cancer. Among the key endocytic regulatory proteins are more than 50 Rab-GTP binding proteins and their effectors that control vesicle transport and fusion events. Over the past five years, we have focused on understanding the role of a novel endocytic regulatory protein family, known as the C-terminal Eps15 Homology Domain proteins (EHD). Despite a growing number of studies demonstrating roles for EHD proteins in the regulation of transport of a variety of receptors, thus far a unifying hypothesis for the actual mechanistic function of EHD proteins has remained elusive. EH-domains interact with proteins containing the tripeptide motif asparagine-proline- phenylalanine (NPF). Most significantly for this renewal application, we have discovered that the protein known as Molecule Interacting with CasL-Like 1 (MICAL-L1) is a novel NPF-containing interaction partner of EHD1. MICAL-L1 acts as unusual Rab effectors because it is critical for the recruitment of both Rab8a and EHD1 to tubular recycling endosomes that are enriched in phosphatidylinositol-4-phosphate and phosphatidylinositol-4, 5-bisphosphate. Depletion of MICAL-L1 simulates EHD1 depletion, causing a delay in recycling to the plasma membrane and an accumulation of internalized receptors at the endocytic recycling compartment (ERC). Moreover, new preliminary data show that MICAL-L1 also interacts with the membrane-bending BAR-domain protein, Syndapin II. Collectively, our data indicate a key role for MICAL-L1 in regulating the generation of tubular endosomes, and in recruiting EHD1, which appears to be responsible for their subsequent vesiculation. Our central hypothesis is that EHD1 plays a critical role in tubular membrane scission and facilitates recycling of membrane and proteins to the plasma membrane. Our first specific aim is to determine the mechanism by which EHD1 functions in tubule membrane scission and recycling. Our working hypothesis is that ATP hydrolysis by EHD1 promotes scission of vesicles from EHD1- containing tubules, thus supporting recycling to the plasma membrane. Our second Specific Aim is to characterize the roles of select EHD1 interaction partners in tubule membrane generation and in the modulation of EHD1 function. We hypothesize that BAR-domain-containing EHD1 interaction partners (such as Syndapin II and/or Bin1) generate the tubular membranes to which EHD1 is recruited. We further hypothesize that MICAL-L1 plays a crucial role in regulating EHD1 function by maintaining it on tubular membranes. Ultimately, these studies will facilitate the development of new strategies to treat the many diseases that arise as a result of aberrant endocytic events.