Polycystin2 function in zebrafish and medaka

描述（由申请人提供）：常染色体多囊肾病（ADPKD）是由编码多囊蛋白1 （PC1）和多囊蛋白2 （PC2）的PKD1和PKD2基因突变引起的。PC2是一种非选择性Ca2+可渗透通道，可在多个亚细胞位置（质膜、纤毛、内质网和高尔基体）发挥作用，但调控PC2在不同细胞间动态易位的因素尚不清楚。鉴于很难在人类或小鼠中研究PC2的功能和信号传导，需要替代动物模型来验证体外研究的数据。该基金的目标是建立人类PC2在斑马鱼和medaka中的动态易位机制，确定PC2在天然细胞环境中生理上重要的蛋白伴侣，并研究调节PC2功能的可能的信号转导途径。我们已经证明PC2 Ser76被糖原合成酶激酶3 （GSK3）磷酸化。在GSK3抑制剂存在的情况下，内源性PC2的侧质膜池重新分布到MDCK细胞的细胞内腔室，但仍与初级纤毛相关。共同注射野生型，而不是S76A/S80A突变型的人PKD2 mRNA，可以修复由破坏内源性斑马鱼PKD2基因引起的缺陷。我们得出结论，PC2的定位在体内和体外都受到一个独特的GSK3磷酸化位点的调节。基于这些数据，我们假设除了纤毛外，PC2功能还需要靶向特定的亚细胞结构域。我们的具体目标是：1。利用斑马鱼和medaka鉴定正确亚细胞靶向和正常PC2功能所需的人类PC2功能基序；2. 确定两个候选pc2相互作用蛋白PIGEA14和PACS和3的功能。以确定WNT、BMP依赖通路是PC2亚细胞靶向所必需的。PC2在人类、斑马鱼和medaka中功能保守。因此，这些研究将有助于阐明PC2在正常人肾脏和病理疾病阶段的功能。这些数据将定义调控PC2亚细胞定位和功能的途径，并确定新的治疗靶点。

DESCRIPTION (provided by applicant): Autosomal Polycystic Kidney Disease (ADPKD) is caused by mutations in PKD1 and PKD2 gene that encode polycystin 1 (PC1) and polycystin2 (PC2). PC2 is a non-selective Ca2+permeable channel, which may function in more than one subcellular location (plasma membrane, cilia, ER and Golgi), but the factors that regulate the dynamic translocation of PC2 between different cellular compartments are not well understood. Given that it is difficult to study PC2 function and signaling in humans or mice, alternative animal models are necessary to validate data derived from in vitro studies. The goal of this grant is to establish mechanisms for the dynamic translocation of human PC2 in zebrafish and medaka, to identify physiologically important protein partners of PC2 in the native cellular environment and to investigate possible signal transduction pathways regulating PC2 function. We have shown that PC2 Ser76 is phosphorylated by glycogen synthase kinase 3 (GSK3). In the presence of GSK3 inhibitors, the lateral plasma membrane pool of endogenous PC2 redistributes into an intracellular compartment of MDCK cells but remains associated with the primary cilium. Co-injection of wild-type, but not a S76A/S80A mutant human PKD2 mRNA, rescued defects induced by disrupting the endogenous zebrafish pkd2 gene. We conclude that localization of PC2 is regulated by a unique GSK3 phosphorylation site both in vivo and in vitro. Based on this data, we hypothesize that PC2 function requires targeting to specific subcellular domains in addition to the cilium. Our specific aims are: 1. To use zebrafish and medaka to identify human PC2 functional motifs required for correct subcellular targeting and normal PC2 function; 2. to define function of two candidate PC2-interacting proteins PIGEA14 and PACS and 3. to establish that WNT, BMP dependent pathways are required for subcellular targeting of PC2. PC2 is functionally conserved among human, zebrafish and medaka. Therefore these studies will help elucidate PC2 function in normal human kidney and in pathological disease stages. These data will define pathways that regulate PC2 subcellular localization and function and identify novel therapeutic targets.