Polycystin2 function in zebrafish and medaka

常染色体多囊肾病（ADPKD）是由PKD 1和PKD 2基因突变引起的， 编码多囊蛋白1（PC 1）和多囊蛋白2（PC 2）。PC 2是一种非选择性的Ca 2+渗透通道， 可能在一个以上的亚细胞位置（质膜，纤毛，ER和高尔基体）发挥作用，但这些因子 调节PC 2在不同细胞区室之间动态移位的机制尚不完善 明白鉴于难以在人类或小鼠中研究PC 2功能和信号传导，替代动物实验室可以在小鼠中进行。 模型对于验证来自体外研究的数据是必要的。该基金的目标是建立 人类PC 2在斑马鱼和青鳉中动态易位的机制，以确定生理学上的 PC 2在天然细胞环境中的重要蛋白伴侣，并研究可能的信号 调节PC 2功能的转导途径。我们已经证明，PC 2 Ser 76被磷酸化， 糖原合成酶激酶3（GSK 3）。在存在GSK 3抑制剂的情况下，侧质膜池 内源性PC 2重新分布到MDCK细胞的细胞内区室，但仍与 与初级纤毛相连共注射野生型而非S76 A/S80 A突变体人PKD 2 mRNA， 通过破坏内源性斑马鱼PKD 2基因诱导的缺陷。我们得出结论，PC 2的定位是 在体内和体外均由独特的GSK 3磷酸化位点调节。根据这些数据，我们 假设PC 2功能需要靶向除纤毛之外的特定亚细胞结构域。我们 具体目标是：1.利用斑马鱼和青鳉来鉴定人类PC 2功能基序， 正确的亚细胞靶向和正常的PC 2功能; 2.定义两个候选PC 2的功能-交互 蛋白质PIGEA 14和PACS和3。为了确定WNT，BMP依赖性途径是 PC 2的亚细胞靶向。PC 2在人类、斑马鱼和青鳉中是功能保守的。因此 这些研究将有助于阐明PC 2在正常人肾脏和病理疾病阶段中的功能。 这些数据将定义调节PC 2亚细胞定位和功能的途径，并识别新的 治疗目标

Autosomal Polycystic Kidney Disease (ADPKD) is caused by mutations in PKD1 and PKD2 gene that encode polycystinl (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 PC2function. 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.