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

描述（由申请人提供）：钠氢交换调节因子-1 （NHERF-1）是一种适配器蛋白，包含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边界膜，并与Npt2a结合，Npt2a是主要的钠依赖性磷酸盐转运蛋白。我们最近的研究表明，NHERF-1-/-肾近端小管细胞中的钠依赖性磷酸盐运输抵抗甲状旁腺激素（PTH）的抑制作用。在当前的应用中，我们探索了PTH介导NHERF-1的PDZ - I特异性残基磷酸化的假设，从而调节Npt2a/NHERF-1复合物，肾近端小管细胞顶膜中Npt2a的丰度，并因此影响磷酸盐的小管重吸收。阐明调控靶蛋白与NHERF-1的PDZ -1结合的因素，也可能提供更广泛的见解，了解接头蛋白PDZ结构域的调控如何影响激素的生物反应和NHERF-1相关疾病的病理生理学。在完整的动物和培养的近端小管细胞中，我们将使用生理、生化和细胞生物学分析来确定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。我们提出三个具体目标。首先，我们将绘制NHERF-1的PDZ I中的丝氨酸和/或苏氨酸残基，这些残基在PTH和下游蛋白激酶的作用下被磷酸化。其次，我们建议通过体外和体内实验研究Npt2a/NHERF-1复合物在pth介导的NHERF-1磷酸化反应中的关联和解离。第三，我们将确定pth介导的NHERF-1磷酸化对肾近端小管中磷酸盐运输的调节的生理作用。

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.