Cytoskeletal linking proteins in renal epithelial cell function

描述（由申请人提供）：在肾近端小管（PT）细胞中，血清磷酸盐调节的关键事件是尖膜处磷酸钠2a （NaPiIIa）共转运蛋白的插入、保留和恢复。NaPiIIa活性受其与许多不同的PDZ蛋白（包括EBP50和PDZK1）的直接相互作用的影响。这些蛋白似乎影响NaPiIIa在顶膜中的传递和保留。最近，一种新的PDZ蛋白Shank2E参与调控NaPiIIa。已知Shank2E结合内吞蛋白（如动力蛋白，内联蛋白），当NaPiIIa从顶膜内吞时，Shank2E结合NaPiIIa并与NaPiIIa一起重新分布到细胞内部。因此，以下提案将验证Shank2E以一种受调节的方式协调NaPiIIa内吞作用的假设。为此，拟制的研究将(1)确定Shank2E与NaPiIIa相关蛋白之间受调节的蛋白-蛋白相互作用，(2)确定Shank2E在近端小管细胞模型中调节NaPiIIa活性的作用，(3)在全动物模型中证明Shank2E在NaPiIIa调节中的作用。这些研究的结果将在三个方面取得重要进展。在分子水平上，这些研究将确定以shank2e为中心的蛋白的修饰-与其他PDZ结构域蛋白和NaPiIIa的蛋白相互作用是如何协调调节的。在细胞和器官水平上，这些研究将阐明这些协调调节的相互作用如何导致Shank2E指导NaPiIIa在生理刺激下的内吞恢复。最后，这些结果可能提供机制洞察到磷血症疾病和指导新的治疗策略的起源。公共卫生相关性：拟议研究的广泛目标是更好地了解身体如何调节盐和水进出身体的运动。肾脏是维持体内盐和水平衡的中心器官。肾脏的磷酸盐平衡对维持身体的健康状态至关重要。磷酸盐水平的长期下降会导致骨骼和牙齿的虚弱和畸形。另外，即使是磷酸盐水平的适度增加也会导致血管沉积和心血管疾病。调节体内磷酸盐平衡的关键事件是肾脏中磷酸盐转运蛋白的活性水平。拟议的研究旨在发现和定义最近在肾脏中发现的一种蛋白质（称为Shank2E）如何能够降低磷酸盐转运蛋白的活性。有趣的是，Shank2E蛋白还能结合其他关键转运蛋白。其中包括与囊性纤维化有关的氯离子通道和与杜宾-约翰逊综合征有关的肝脏胆红素转运蛋白。通过发现Shank2E如何调节这些蛋白质的功能，研究结果阐明了身体如何在正常条件下维持自身，这些蛋白质的变化如何引起特定疾病，以及研究人员如何利用这些调节机制开发治疗这些疾病的医学疗法。

DESCRIPTION (provided by applicant): In renal proximal tubule (PT) cells, the pivotal event in serum phosphate regulation is the insertion, retention and recovery of the sodium-phosphate 2a (NaPiIIa) co-transporter at the apical membrane. NaPiIIa activity is influence by its direct interaction with a number of distinct PDZ proteins, including EBP50 and PDZK1. These proteins appear to influence the delivery and retention of NaPiIIa in the apical membrane. More recently, a novel PDZ protein, Shank2E, was implicated in regulating NaPiIIa. Already known to bind endocytic proteins (e.g. dynamin, cortactin), Shank2E binds NaPiIIa and redistribute with NaPiIIa into the cell interior when NaPiIIa was endocytosed from the apical membrane. Consequently, the following proposal will test the hypothesis that Shank2E coordinates the endocytosis of NaPiIIa in a regulated manner. To this end, the proposed studies will (1) define the regulated protein-protein interactions between Shank2E and NaPiIIaassociated proteins, (2) determine the role of Shank2E in moderating NaPiIIa activity in proximal tubule cell models and (3) demonstrate the role of Shank2E in NaPiIIa regulation in whole animal models. Results from these studies will make important advances on three fronts. At the molecular level, the studies will determine how modifications in Shank2E-centered protein-protein interactions with other PDZ domain proteins and NaPiIIa are coordinately regulated. At the cellular and organ level, the studies will elucidate how these coordinately regulated interactions result in Shank2E directing the endocytic recovery of NaPiIIa in response to physiologic stimuli. Finally, these results may provide mechanistic insight into phosphatemic diseases and direct the genesis of novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: The broad goal of the proposed studies is to better understand how the body regulates the movement of salts and water in and out of the body. The kidney is the central organ involved in maintaining salt and water balance in the body. The balance of phosphate by the kidney is essential for maintaining the health status of the body. Chronic decreases of phosphate levels can result in weaknesses and deformities in bones and teeth. Alternatively, even modest increases in phosphate levels can result in vascular deposits and cardiovascular disease. The pivotal event in regulating the balance of phosphate in the body is the activity level of a phosphate transport protein in the kidney. The proposed studies seek to discover and define how a recently identified protein in the kidney (termed Shank2E) is capable of turning down the activity of the phosphate transport protein. Interestingly, the Shank2E protein also binds other key transport proteins. These include a chloride channel that is linked to Cystic Fibrosis and a bilirubin transport protein in the liver that is linked to Dubin-Johnson Syndrome. By discovering how Shank2E functions to regulate these proteins the findings elucidate how the body maintains itself under normal conditions, how changes in these proteins might give rise to specific diseases and how researchers might take advantage of these regulated mechanisms to develop medical therapies to treat these diseases.