Cytoskeletal linking proteins in renal epithelial cell function

描述(申请人提供)：在肾近端小管(PT)细胞中，血清磷调节的关键事件是在顶膜插入、保留和恢复磷酸钠2a(NaPiIIa)共转运体。NaPiIIa的活性受到其与许多不同的PDZ蛋白的直接相互作用的影响，包括EBP50和PDZK1。这些蛋白质似乎影响了NaPiIIa在顶膜中的传递和保留。最近，一种新的PDZ蛋白Shank2E被发现参与调节NaPiIIa。Shank2E已知可以结合内吞蛋白(如动力素、皮质蛋白)，当NaPiIIa从顶膜被吞噬时，Shank2E与NaPiIIa结合，并与NaPiIIa一起重新分布到细胞内部。因此，以下建议将检验以下假设，即Shank2E以调节的方式协调NaPiIIa的内吞作用。为此，拟议的研究将(1)确定Shank2E和NaPiIIa相关蛋白之间受调控的蛋白质-蛋白质相互作用，(2)确定Shank2E在调节近端小管细胞模型中NaPiIIa活性的作用，以及(3)在整个动物模型中证明Shank2E在NaPiIIa调控中的作用。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为中心的蛋白质-蛋白质与其他PDZ结构域蛋白质和NaPiIIa之间的修饰是如何被协调调控的。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对生理刺激的内吞恢复。最后，这些结果可能提供对磷酸血症性疾病的机械性洞察，并指导新的治疗策略的起源。与公共卫生相关：拟议研究的广泛目标是更好地了解身体如何调节盐分和水在体内和体外的流动。肾脏是维持体内盐分和水分平衡的中心器官。肾脏对磷酸盐的平衡对维持身体的健康状态至关重要。磷酸盐水平的慢性下降会导致骨骼和牙齿的虚弱和畸形。或者，即使磷酸盐水平略有增加，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动水平。拟议中的研究试图发现和定义肾脏中最近发现的一种蛋白质(称为Shank2E)是如何能够抑制磷酸转运蛋白的活性的。有趣的是，Shank2E蛋白还与其他关键运输蛋白结合。其中包括与囊性纤维化有关的氯离子通道，以及与杜宾-约翰逊综合征有关的肝脏中的胆红素转运蛋白。通过发现Shank2E如何调节这些蛋白质的功能，这些发现阐明了身体在正常条件下如何维持自身，这些蛋白质的变化可能如何导致特定的疾病，以及研究人员如何利用这些调节机制来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.