Role of GRP170 in ENaC Biogenesis and Renal Physiology

项目摘要 这项建议的重点是研究保守的分子伴侣， GRP 170/Lhs 1调节上皮钠通道ENaC的降解、组装和运输。 ENaC负责肾脏和肺上皮细胞的盐重吸收，并控制血液和肺组织。 压力、离子和体液平衡。ENaC的功能获得和丧失突变导致疾病， ENaC活性还与其他与上皮功能障碍相关的疾病相关。ENaC是一个 由α、β和γ亚基组成的异源三聚体通道。每个亚基含有两个跨膜结构域， 一个大的细胞外环，和短的胞质N-和C-末端。合成后不久，ENaC受到 内质网相关降解（ERAD），靶向错误折叠的蛋白质和孤儿 细胞质26 S蛋白酶体破坏的多聚体复合物的亚基。毫不奇怪，ENaC 亚基单独作为ERAD的目标，但即使所有三个亚基都被降解， ENaC亚基存在。如何足够的亚基组装发生，以逃避ERAD是神秘的。 然而，来自这个研究小组的数据揭示了Lhs 1分子伴侣的新作用（GRP 170在 哺乳动物细胞）。具体来说，Lhs 1促进α亚基的降解，但 对β或γ亚基降解没有影响，但当所有三种ENaC亚基表达时，亚基间 跨膜结构域之间的相互作用阻断了Lhs 1依赖的ERAD。与这些数据一致， GRP 170还靶向哺乳动物细胞中ERAD的α亚基，但促进组装的 异源三聚体通道三个模型系统将被用来进一步理解这些事件：1）一个既定的， 遗传上容易的酵母系统将被用来定义区分一种 孤儿ENaC亚基和组装的异源三聚体通道; 2）Fischer大鼠甲状腺（FRT）细胞系统 将用于确认酵母系统的结果，并确定GRP 170介导的 3）条件性GRP 170敲除小鼠，其在肾脏中缺乏GRP 170 肾小管，将用于确定GRP 170伴侣蛋白如何调节ENaC影响肾脏生理学。 总的来说，这项建议将使用多系统的方法来定义一个单一的分子伴侣如何调节 ENaC和-第一次-表明伴侣如何选择一个孤儿亚基降解以及 以促进寡聚蛋白质的组装。总之，了解GRP 170的作用机制将有助于 为ENaC功能和相关疾病状态提供了新的见解。更一般地说，这项工作将有助于 破译ER中多聚体蛋白的膜组装如何导致稳定和运输， 对于肾脏中许多其他离子转运蛋白的功能至关重要。本文中描述的实验 一个多学科调查员小组将与当地专家合作，为这项建议提供便利 ENaC生理学、成像技术、鼠疾病模型和ERAD。

Project Summary The focus of this proposal is to investigate the mechanism by which the conserved molecular chaperone, GRP170/Lhs1, regulates the degradation, assembly, and trafficking of the epithelial sodium channel, ENaC. ENaC is responsible for salt reabsorption across epithelia of the kidney and lung, and controls both blood pressure and ion and fluid homeostasis. Gain- and loss-of-function mutations in ENaC lead to disease, and ENaC activity is also associated with other diseases associated with epithelial malfunction. ENaC is a heterotrimeric channel composed of an α, β, and γ subunit. Each subunit contains two transmembrane domains, a large extracellular loop, and short cytosolic N- and C-termini. Soon after synthesis, ENaC is subject to Endoplasmic Reticulum Associated Degradation (ERAD), which targets misfolded proteins and orphaned subunits of multimeric complexes for destruction by the cytosolic 26S proteasome. Not surprisingly, ENaC subunits individually are targeted for ERAD, but a significant percent of ENaC is degraded even when all three ENaC subunits are present. How sufficient subunit assembly occurs in order to escape ERAD is mysterious. However, data from this team of investigators uncovered a new role for the Lhs1 chaperone (GRP170 in mammalian cells) during ENaC biogenesis. Specifically, Lhs1 facilitated the degradation of the α subunit but had no effect on β or γ subunit degradation, yet when all three ENaC subunits were expressed, intersubunit interactions between the transmembrane domains blocked Lhs1-dependent ERAD. Consistent with these data, GRP170 also targeted the α subunit for ERAD in mammalian cells but promoted trafficking of the assembled heterotrimeric channel. Three model systems will be used to further understand these events: 1) An established, genetically facile yeast system will be used to define the structural elements required to differentiate between an orphaned ENaC subunit and the assembled heterotrimeric channel; 2) A Fischer rat thyroid (FRT) cell system will be used to confirm results from the yeast system and define amino acid motifs required for GRP170-mediated channel assembly and trafficking; 3) A conditional GRP170 knock out mouse, which lacks GRP170 in kidney tubules, will be used to determine how ENaC regulation by the GRP170 chaperone affects renal physiology. Overall, this proposal will use a multi-system approach to define how a single molecular chaperone regulates ENaC and—for the first time—indicate how chaperones can select an orphaned subunit for degradation as well as facilitate assembly of an oligomeric protein. Together, understanding the mechanism of action of GRP170 will provide novel insights into ENaC function and associated disease states. More generally, this work will help decipher how membrane assembly of a multimeric protein in the ER results in stabilization and trafficking, which is vital for the function of numerous other ion transporters in the kidney. The experiments described in this proposal will be facilitated by a multi-disciplinary team of investigators and by collaborations with local experts in ENaC physiology, imaging technologies, murine disease models and ERAD.