Regulation of polycystin-2 channel activity

多囊蛋白-2 通道活性的调节

基本信息

批准号：
8320422
负责人：
Titus Jonathon Boggon
金额：
$ 39.33万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8320422
关键词：
Address Affect Affinity Agonist Amino Acid Sequence Amino Acids Animals Area Binding Biochemical C-terminal Calcium Cations Cells Cilia Coiled-Coil Domain Complex Cyst Cystic kidney Data Disease Dyes EF Hand Motifs EF-Hand Domain End stage renal failure Etiology Extracellular Domain Family Genes Goals Hepatic Cyst Hereditary Disease Imaging Techniques In Vitro Individual Inherited Kidney Lead Left Length Measurement Mediating Molecular Molecular Structure Monitor Mus Mutate Mutation NMR Spectroscopy Nuclear Magnetic Resonance PKD2 gene PKD2 protein Phenotype Phosphorylation Physiological Polycystic Kidney Diseases Proteins Regulation Renal function Roentgen Rays Role Serine Side Signal Transduction Structure Tail Techniques Transfection Transgenic Mice analytical ultracentrifugation design disease phenotype falls fluid flow improved in vivo in vivo Model mutant polycystic kidney disease 1 protein public health relevance receptor response three dimensional structure

项目摘要

DESCRIPTION (provided by applicant): Polycystic kidney disease (PKD) is a systemic hereditary disease characterized by renal and hepatic cysts. There is no known cure for this disease and it results in end-stage renal failure in approximately 50% of affected individuals. Mutations in either the PKD1 or the PKD2 gene, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, are seen in over 95% of PKD cases. An inability of PC1 or PC2 to function normally is highly correlated with cystogenesis in PKD. There are currently, however, serious deficiencies in the molecular-level descriptions of PC1 and PC2, and in understanding how alterations in these primary amino acid sequences affect three-dimensional structure, in vitro function and in vivo phenotypes. Our plan is to use a multi-disciplinary approach to significantly improve the description of PC2. We will conduct studies that fall along a structure-function-phenotype axis that will describe the consequences of PC2 mutation in PKD. In Aim 1 we will concentrate on describing the role Ca2+ binding to the EF-hand motif in the C-terminal cytoplasmic region of PC2. This region is critical for normal PC2 Ca2+ channel function and is often truncated in PKD. We will determine the structure of the C-terminal cytoplasmic region of PC2 in the presence of Ca2+ using NMR techniques. This structure will allow us to rationally design mutations we expect to alter Ca2+ binding and therefore PC2 channel function. We will then investigate the functional relevance of this region to PC2 Ca2+ signaling by conducting single channel measurements of PC2 channels in bilayers and by monitoring intracellular Ca2+ changes after agonist addition. In Aim 2 we will investigate the role of phosphorylation of PC2 at serine 812, a residue located between the EF-hand and coiled-coil domains of PC2 that has been shown to be important for channel regulation and is often disrupted in PKD. We will investigate the effect of phosphorylation mimics on Ca2+ binding, on the global structure of the PC2 C-terminal tail, and on the direct C- terminal tail mediated PC1-PC2 interaction. The functional consequences of phosphorylation will be studied using single channel techniques and imaging of fluorescent Ca2+-sensitive dyes in intact cells. In Aim 3 we will investigate the functional consequences of the interactions described in aims 1 and 2, the roles of Ca2+ binding and phosphorylation of PC2. For wild-type and mutant PC2 we will examine flow induced changes in Ca2+ signals, in cells obtained by transfection and in cells obtained by isolation from transgenic mice. We will generate transgenic mice that express wild-type or mutant PC2 and examine the hallmarks of PKD, the right- left axis and cyst formation. The studies proposed will allow us to correlate atomic-level molecular structures of PC2 with biophysical functional analyses and PKD phenotypes in mice. Or results will significantly enhance our molecular, functional and physiological understanding of PC2 in normal kidney function and the consequences of their dysregulation in PKD. PUBLIC HEALTH RELEVANCE: The project aims to enhance our molecular, functional and physiological understanding of the two proteins most often mutated in polycystic kidney disease, polycystin-1 and polycystin-2. We will determine the structure of a region important for regulation of this complex, will conduct functional studies and will describe the effects of mutations in mice for both polycystin-2. The proposed studies will help us understand the role of polycystin-1 and polycystin-2 in normal kidneys and in those affected by polycystic kidney disease.

描述（申请人提供）：多囊肾病（PKD）是一种以肾脏和肝脏囊肿为特征的全身性遗传性疾病。这种疾病尚无已知的治愈方法，大约 50% 的受影响个体会导致终末期肾衰竭。超过 95% 的 PKD 病例中可观察到分别编码多囊蛋白-1 (PC1) 和多囊蛋白-2 (PC2) 的 PKD1 或 PKD2 基因突变。 PC1 或 PC2 无法正常发挥功能与 PKD 中的囊肿发生高度相关。然而，目前对 PC1 和 PC2 的分子水平描述以及对这些一级氨基酸序列的改变如何影响三维结构、体外功能和体内表型的理解还存在严重缺陷。我们的计划是使用多学科方法来显着改进 PC2 的描述。我们将沿着结构-功能-表型轴进行研究，描述 PC2 突变对 PKD 的影响。在目标 1 中，我们将集中描述 Ca2+ 与 PC2 C 末端细胞质区域中 EF-hand 基序结合的作用。该区域对于正常 PC2 Ca2+ 通道功能至关重要，并且在 PKD 中经常被截断。我们将使用 NMR 技术确定 Ca2+ 存在下 PC2 C 末端细胞质区域的结构。这种结构将使我们能够合理地设计突变，我们期望改变 Ca2+ 结合，从而改变 PC2 通道功能。然后，我们将通过对双层 PC2 通道进行单通道测量并监测添加激动剂后细胞内 Ca2+ 的变化，研究该区域与 PC2 Ca2+ 信号传导的功能相关性。在目标 2 中，我们将研究 PC2 丝氨酸 812 磷酸化的作用，丝氨酸 812 是位于 PC2 EF 手和卷曲螺旋结构域之间的残基，已被证明对通道调节很重要，并且在 PKD 中经常被破坏。我们将研究磷酸化模拟物对 Ca2+ 结合、PC2 C 末端尾部整体结构以及直接 C 末端尾部介导的 PC1-PC2 相互作用的影响。将使用单通道技术和完整细胞中荧光 Ca2+ 敏感染料的成像来研究磷酸化的功能后果。在目标 3 中，我们将研究目标 1 和 2 中描述的相互作用的功能后果、Ca2+ 结合和 PC2 磷酸化的作用。对于野生型和突变型 PC2，我们将检查流诱导的 Ca2+ 信号、转染获得的细胞和转基因小鼠分离获得的细胞中的变化。我们将培育表达野生型或突变型 PC2 的转基因小鼠，并检查 PKD 的特征、左右轴和囊肿形成。拟议的研究将使我们能够将 PC2 的原子级分子结构与小鼠的生物物理功能分析和 PKD 表型联系起来。或者结果将显着增强我们对正常肾功能中的 PC2 及其在 PKD 中失调的后果的分子、功能和生理学理解。公共健康相关性：该项目旨在增强我们对多囊肾病中最常突变的两种蛋白质（多囊蛋白-1 和多囊蛋白-2）的分子、功能和生理学了解。我们将确定对该复合物调节重要的区域的结构，将进行功能研究，并描述小鼠中多囊蛋白-2 突变的影响。拟议的研究将帮助我们了解多囊蛋白-1 和多囊蛋白-2 在正常肾脏和多囊肾病患者中的作用。