Polycystin2 function in zebrafish and medaka

描述(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是一种非选择性的钙离子通透通道，可作用于多个亚细胞位置(质膜、纤毛、内质网和高尔基体)，但调节PC2在不同细胞间动态转运的因素尚不清楚。考虑到很难研究PC2在人类或小鼠中的功能和信号，有必要建立替代的动物模型来验证来自体外研究的数据。这笔赠款的目的是建立人PC2在斑马鱼和青竹中动态易位的机制，确定PC2在自然细胞环境中的重要生理蛋白伙伴，并研究调节PC2功能的可能信号转导途径。我们已经证明PC2Ser76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，但仍与初级纤毛有关。共同注射野生型，而不是S76A/S80A突变的人PKD2基因，可以修复由于破坏内源性斑马鱼PKD2基因而导致的缺陷。我们得出结论，在体内和体外，PC2的定位都受到独特的GSK3磷酸化位点的调控。基于这些数据，我们假设PC2功能除了纤毛外，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鉴定正确的亚细胞靶向和正常的PC2功能所需的人PC2功能基序；2.确定两个候选PC2相互作用蛋白PIGEA14和PACS的功能；3.建立PC2亚细胞靶向所需的WNT、BMP依赖的通路。PC2在人类、斑马鱼和青蛙中功能保守。因此，这些研究将有助于阐明PC2在正常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.