Polycystin2 function in zebrafish and medaka

多囊蛋白2在斑马鱼和青鳉鱼中的功能

基本信息

批准号：
8102185
负责人：
TOMOKO OBARA
金额：
$ 25.13万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8102185
关键词：
Accounting Affect Animal Model Animals Anterior Antibodies Autosomal Dominant Polycystic Kidney Biological Models Cell Culture Techniques Cell membrane Cell physiology Cilia Complementary DNA Cyst Cystic Kidney Diseases Data Defect Disease Disease model Dominant-Negative Mutation Embryo Endoplasmic Reticulum Environment Etiology Fishes Gene Mutation Gene Proteins Genes Genetic Screening Glycogen (Starch) Synthase Glycogen Synthase Kinase 3 Goals Golgi Apparatus Grant Heat-Shock Response Homologous Gene Human In Vitro Inbreeding Injection of therapeutic agent Kidney Larva Lateral Lead Length Letters Life Link Location MDCK cell Mediating Messenger RNA Modeling Monitor Mus Mutation Names Nomenclature PKD1 gene PKD2 gene Pathway interactions Phenotype Phosphorylation Site Play Polycystic Kidney Diseases Proteins Publishing Regulation Research Personnel Role Signal Pathway Signal Transduction Signal Transduction Pathway Site-Directed Mutagenesis Sorting - Cell Movement Staging System TCF3 gene Testing Therapeutic Agents Tissues Transgenic Organisms Zebrafish base cellular targeting in vivo inhibitor/antagonist molecular mass mutant new therapeutic target novel polycystic kidney disease 1 protein programs promoter receptor research study trafficking

项目摘要

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.

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 Ca2+ 通透通道，可能在多个亚细胞位置（质膜、纤毛、内质网和高尔基体）发挥作用，但调节 PC2 在不同细胞区室之间动态易位的因素尚不清楚。鉴于在人类或小鼠中研究 PC2 功能和信号传导很困难，因此需要替代动物模型来验证体外研究得出的数据。该资助的目标是建立斑马鱼和青鳉中人类 PC2 动态易位的机制，识别天然细胞环境中 PC2 的生理上重要的蛋白质伴侣，并研究调节 PC2 功能的可能信号转导途径。我们已经证明 PC2 Ser76 被糖原合酶激酶 3 (GSK3) 磷酸化。在 GSK3 抑制剂存在的情况下，内源性 PC2 的侧质膜库重新分布到 MDCK 细胞的细胞内区室中，但仍与初级纤毛相关。共注射野生型而非 S76A/S80A 突变型人 PKD2 mRNA，可以挽救因破坏内源性斑马鱼 pkd2 基因而引起的缺陷。我们得出结论，PC2 的定位在体内和体外均受到独特的 GSK3 磷酸化位点的调节。基于这些数据，我们假设 PC2 功能需要靶向除纤毛之外的特定亚细胞结构域。我们的具体目标是： 1. 利用斑马鱼和青鳉鱼来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. 定义两种候选 PC2 相互作用蛋白 PIGEA14 和 PACS 的功能，以及 3. 确定 PC2 亚细胞靶向需要 WNT、BMP 依赖性途径。 PC2 在人类、斑马鱼和青鳉鱼中的功能是保守的。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。这些数据将定义调节 PC2 亚细胞定位和功能的途径，并确定新的治疗靶点。