NHERF-1 and dopamine-mediated regulation of renal phosphate transport.

肾小管对磷酸盐的重吸收在很大程度上是由钠- 依赖的磷酸转运蛋白Npt2a位于近曲小管细胞的顶膜上。Npt2a通过我们实验室分离和克隆的一个PDZ结构域适配子蛋白-1(NHERF-1)与顶膜相连。NHERF-1基因缺失的小鼠表现出与肾乳头钙沉积相关的磷酸盐、钙和尿酸排泄增加。在小鼠身上证实，人类的NHERF-1突变与肾结石有关。我们实验室最近的研究表明，甲状旁腺激素导致NHERF-1的第一个PDZ结构域的磷酸化，随后Npt2a/NHERF-1复合体解离，Npt2a的顶膜丰度降低，磷酸盐转运减少。我们推测，这一独特的机制可能是调节NHERF-1与其他一些靶蛋白结合的一般过程。为了研究NHERF-1在肾脏磷酸盐转运中的作用，并确定NHERF-1的磷酸化是否受其他激素的调节，我们提出了研究多巴胺抑制肾脏磷酸盐转运的机制。使用来自野生型和NHERF-1缺失动物的新鲜准备的肾脏切片和原代培养的肾近端小管细胞，我们将首先使用免疫沉淀和共聚焦显微镜确定NHERF-1是否与多巴胺D1样受体结合。使用32P标记的细胞，然后我们将确定多巴胺D1样受体的占据是否导致NHERF-1的磷酸化，并使用腺病毒介导的野生型和突变型NHERF-1的基因转移，确定第一个PDZ结构域(Npt2a的结合部位)的哪些残基被磷酸化。具体的兴趣将集中在位于NHERF-1的Npt2a结合槽中的丝氨酸77上。然后我们将确定 通过测量野生型和NHERF-1缺失细胞中cAMP的积累和PKC活性，多巴胺D1样受体使用的信号通路。为了确定NHERF-1的生理作用，我们将测量多巴胺对野生型和NHERF-1缺失的近端小管细胞钠依赖的磷酸盐运输的影响。还将在感染了腺病毒NHERF-1的NHERF-1缺失细胞中进行研究，以确定NHERF-1在介导多巴胺效应中的特定作用，并与NHERF-1的突变形式一起确定特定残基(如丝氨酸77)的作用。为了研究抑制机制，我们将使用定量免疫沉淀、共聚焦显微镜和蔗糖密度梯度超速离心法确定多巴胺是否解离Npt2a/NHERF-1复合体。因此，我们的直接目标是测试NHERF-1与多巴胺受体(S)相互作用的假设，以及多巴胺通过磷酸化Npt2a结合蛋白来抑制肾脏磷酸盐运输的假设 NHERF-1因此解离NHERF-1/Npt2a复合体并降低心尖部 Npt2a的膜丰度。我们预计荷尔蒙转导的比较 这些途径将为NHERF-1在磷酸盐运输中的生物学作用提供新的见解，并进一步了解NHERF-1在其他生理和病理生理过程中的作用，如肾结石、精神分裂症、牛皮癣、侵袭性雌激素受体阳性乳腺癌和神经性耳聋(Usher综合征)。 。

The renal tubular reabsorption of phosphate is mediated, in large measure, by a sodium- dependent phosphate transporter called Npt2a located in the apical membrane of the cells of the proximal convoluted tubule. Npt2a is tethered to the apical membrane by a PDZ domain adaptor protein isolated and cloned by our laboratory called the Sodium-Hydrogen Exchanger Regulatory Factor-1 (NHERF-1). NHERF-1 null mice demonstrate increased excretion of phosphate, calcium, and uric acid associated with calcium deposition in the renal papilla. In confirmation with the mouse, NHERF-1 mutations in humans have been associated kidney stones. Recent studies from our laboratory have indicated that Parathyroid hormone results in the phosphorylation of the first PDZ domain of NHERF-1 with the subsequent dissociation of Npt2a/NHERF-1 complexes, a decrease in the apical membrane abundance of Npt2a, and a decrease in phosphate transport. We speculate that this unique mechanism may be a general process that regulates NHERF-1 binding to some of its other target proteins. To study the role of NHERF-1 in renal phosphate transport and to determine if the phosphorylation of NHERF-1 is regulated by other hormones, we propose studies to examine the mechanism by which dopamine inhibits renal phosphate transport. Using freshly prepared kidney slices and renal proximal tubule cells in primary culture from wild-type and NHERF-1 null animals, we will first determine if NHERF-1 binds to the dopamine D1-like receptor using immunoprecipitation and confocal microscopy. Using 32P labeled cells, we will then determine if occupancy of the dopamine D1-like receptor results in the phosphorylation of NHERF-1 and, using adenovirus-mediated gene transfer of wild-type and mutated forms of NHERF-1, determine which residues in the first PDZ domain, the binding site of Npt2a, are phosphorylated. Specific interest will focus on serine77 that lies in the Npt2a binding groove of NHERF-1. We will then determine the signaling pathways used by the dopamine D1-like receptor by measuring cAMP accumulation, and PKC activity in wild-type and NHERF-1 null cells. To determine the physiologic role of NHERF-1, we will measure the effect of dopamine on sodium-dependent phosphate transport in wild-type and NHERF-1 null proximal tubule cells. Studies will also be performed in NHERF-1 null cells infected with adenovirus-NHERF-1 to determine the specific role of NHERF-1 in mediating the effect of dopamine and with mutant forms of NHERF-1 to determine the role of specific residues such as serine77. To examine the mechanism of inhibition, we will determine if dopamine dissociates Npt2a/NHERF-1 complexes using quantitative immunoprecipitation, confocal microscopy, and sucrose density gradient ultracentrifugation. Thus, our immediate objectives are to test the hypothesis that NHERF-1 interacts with the dopamine receptor(s) and that dopamine inhibits renal phosphate transport by phosphorylating the Npt2a binding protein NHERF-1 thereby dissociating NHERF-1/Npt2a complexes and decreasing the apical membrane abundance of Npt2a. We anticipate that comparison of hormonal transduction pathways will provide new insights into the biologic role of NHERF-1 on phosphate transport and, further understandings of the role of NHERF-1 in other physiologic and pathophysiologic processes such as nephrolithiasis, schizophrenia, psoriasis, aggressive forms of estrogen receptor positive breast cancer, and nerve deafness (Usher's syndrome). .