NHERF-1 and PTH regulation of the renal transport of phosphate.

描述(申请人提供)：钠氢交换调节因子-1(NHERF-1)是一种接头蛋白，含有两个蛋白相互作用的PDZ结构域和一个C端ERM结合结构域，定位于肾近曲小管细胞的刷状缘膜，并与主要的钠依赖磷酸转运体Npt2a结合。我们最近的研究表明，NHERF-1/-肾脏近端小管细胞钠依赖的磷酸盐转运对甲状旁腺激素(PTH)的抑制作用具有抵抗力。在目前的应用中，我们探索了这样的假设，即PTH介导NHERF-1的PDZ I特定残基的磷酸化，从而调节Npt2a/NHERF-1复合体，调节Npt2a在肾近端小管细胞顶膜上的丰度，从而调节肾小管对磷酸盐的重吸收。阐明调节靶蛋白与NHERF-1的PDZ I结合的因素也可能为调节适配蛋白的PDZ结构域如何影响激素的生物反应以及NHERF-1相关疾病的病理生理提供更广泛的见解。在完整的动物和培养的近端小管细胞中，我们将使用生理、生化和细胞生物学的方法来确定PTH介导的NHERF-1的PDZ I磷酸化如何调节靶蛋白如Npt2a的结合亲和力和近端小管对磷酸盐的重吸收。我们提出了三个具体目标。首先，我们将定位NHERF-1的PDZ I中的丝氨酸和/或苏氨酸残基，这些残基是PTH和下游蛋白激酶的磷酸化产物。其次，我们建议使用体外和体内实验来研究Npt2a/NHERF-1复合体在PTH介导的NHERF-1磷酸化反应中的结合和解离。第三，我们将确定PTH介导的NHERF-1磷酸化在调节肾脏近端小管磷酸盐运输中的生理学作用。 公共卫生相关性 NHERF-1是一种接头蛋白，可以结合肾脏中的多个转运蛋白，包括主要的肾脏近端小管磷酸转运蛋白Npt2a。我们将研究一种假说，即甲状旁腺激素介导的肾脏磷酸盐转运的调节包括通过NHERF-1的PDZ I结构域的特异性磷酸化来调节Npt2a与NHERF-1的结合。这些观察结果可能为调节靶蛋白与接头(如NHERF-1)结合的过程提供机械性见解，并为NHERF-1相关疾病的病理生理学提供更广泛的见解。

DESCRIPTION (provided by applicant): The Sodium-Hydrogen Exchanger Regulatory Factor-1 (NHERF-1) is an adaptor protein containing two protein-interactive PDZ domains and a C-terminal ERM binding domain that localizes to the brush border membrane of renal proximal convoluted tubule cells and binds to Npt2a, the major sodium-dependent phosphate transporter. Our recent studies have indicated that sodium-dependent phosphate transport in proximal tubule cells from NHERF-1-/- kidneys are resistant to the inhibitory effect of Parathyroid Hormone (PTH). In this current application, we explore the hypothesis that PTH mediates the phosphorylation of specific residues in PDZ I of NHERF-1 thereby regulating Npt2a/NHERF-1 complexes, the abundance of Npt2a in the apical membrane of renal proximal tubule cells, and as a consequence, the tubular reabsorption of phosphate. Elucidation of the factors that regulate the binding of target proteins to PDZ I of NHERF-1 may also provide broader insights into how regulation of PDZ domains of adaptor proteins impact on biologic responses to hormones and on the pathophysiology of NHERF-1 related diseases. In intact animals and cultured proximal tubule cells, we will use physiologic, biochemical, and cell biologic assays to determine how PTH-mediated phosphorylation of PDZ I of NHERF-1 regulateS the binding affinity of target proteins such as Npt2a and the proximal tubule reabsorption of phosphate. We propose three specific aims. First, we will map the serine and/or threonine residues in PDZ I of NHERF-1 that are phosphorylated in response to PTH and downstream protein kinases. Second, we propose to study the association and dissociation of Npt2a/NHERF-1 complexes in response to PTH-mediated phosphorylation of NHERF-1 using in-vitro and in-vivo assays. Third, we will determine the physiologic role of PTH-mediated NHERF-1 phosphorylation on the regulation of phosphate transport in the proximal tubule of the kidney. PUBLIC HEALTH RELEVANCE NHERF-1 is an adaptor protein that binds multiple transporters in the kidney including Npt2a, the major renal proximal tubule phosphate transporter. We will study the hypothesis that Parathyroid Hormone-mediated regulation of renal phosphate transport involves regulation of the binding of Npt2a to NHERF-1 by site-specific phosphorylation of the PDZ I domain of NHERF-1. These observations may provide mechanistic insights into the processes that regulate the binding of target proteins to adaptors such as NHERF-1 and provide broader insights into the pathophysiology of NHERF-1 related diseases.