Polycystin2 function in zebrafish and medaka

多囊蛋白2在斑马鱼和青鳉鱼中的功能

• 批准号: 7877956
• 负责人: TOMOKO OBARA
• 金额: $ 25.38万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7877956
• 关键词: Accounting; Affect; Animal Model; Animals; Anterior; Antibodies; Autosomal Dominant Polycystic Kidney; Biological Models; Cell Culture Techniques; Cell membrane; Cilia; 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; Injection of therapeutic agent; Kidney; Larva; Lateral; Lead; Length; Letters; Life; Link; Location; MDCK cell; Mediating; Messenger RNA; Modeling; Monitor; Mus; Mutation; Names; Nomenclature; 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

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（由申请方提供）：常染色体多囊肾病（ADPKD）是由编码多囊蛋白1（PC 1）和多囊蛋白2（PC 2）的PKD 1和PKD 2基因突变引起的。PC 2是一种非选择性的Ca 2+渗透通道，可能在多个亚细胞位置（质膜、纤毛、ER和高尔基体）发挥作用，但调节PC 2在不同细胞区室之间动态易位的因素还不清楚。鉴于很难在人类或小鼠中研究PC 2的功能和信号传导，需要替代动物模型来验证来自体外研究的数据。这项资助的目标是建立人类PC 2在斑马鱼和青鳉中动态易位的机制，以确定PC 2在天然细胞环境中的生理学重要蛋白质伴侣，并研究可能的信号转导途径调节PC 2功能。我们已经表明，PC 2 Ser 76是磷酸化的糖原合成酶激酶3（GSK 3）。在GSK 3抑制剂的存在下，内源性PC 2的侧质膜池重新分配到MDCK细胞的细胞内隔室中，但仍与初级纤毛相关。共注射野生型，而不是S76 A/S80 A突变体人PKD 2 mRNA，挽救了破坏内源性斑马鱼PKD 2基因诱导的缺陷。我们的结论是，本地化的PC 2是由一个独特的GSK 3磷酸化位点在体内和体外调节。基于这些数据，我们假设PC 2的功能需要靶向特定的亚细胞结构域，除了纤毛。我们的具体目标是：1.利用斑马鱼和青鳉鉴定正确的亚细胞靶向和正常的PC 2功能所需的人PC 2功能基序; 2.确定两个候选的PC 2相互作用蛋白PIGEA 14和PACS的功能;为了确定WNT、BMP依赖性途径是PC 2的亚细胞靶向所必需的。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，并确定新的治疗靶点。