Regulation of Ca++ signaling by the PDK 2 gene product

PDK 2 基因产物对 Ca 信号传导的调节

基本信息

批准号：
6940582
负责人：
Leonidas Tsiokas
金额：
$ 3.51万
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：
2002
资助国家：
美国
起止时间：
2002-03-01 至 2006-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6940582
关键词：
biological signal transduction calcium channel cyclic AMP epithelium gene mutation laboratory mouse polycystic kidney protein structure function tissue /cell culture transfection

项目摘要

DESCRIPTION (provided by applicant): The function(s) of the genes responsible for the vast majority of cases of autosomal dominant polycystic kidney disease (ADPKD) are unknown. Based on sequence analysis, the gene product of the polycystic kidney disease gene 1 (PKD1) has been proposed to encode a protein with a role in cell-cell and/or cell - extracellular matrix interactions while PKD2 is thought to function as a cation channel. We have shown that PKD2 physically associated with PKD1 and only in the presence of PKD1, PKD2 was able to form a Ca++ permeable cation channel. In addition to PKD 1, PKD2 was also able to associate with the transient receptor potential channel 1 (TRPC1). We now show that TRPC1 has a widespread distribution in epithelial structures, primarily the ductal cells of the kidney and liver. TRPC 1 was shown to enhance Ca++ entry in response to store depletion (or capacitative Ca++ entry, CCE). CCE is the major route by which non-excitable cells regulate their intracellular Ca++ concentration. Among the many cellular functions regulated by CCE, regulation of cAMP accumulation is a well-characterized and specific physiological target of CCE. Notably, the involvement of cAMP in cyst formation in kidney and liver epithelial cells has been well established. We propose that PKD1, PKD2 and TRPC1 assemble to a functional complex to enhance CCE. Naturally occurring mutations in PKD2 may result in the disruption of this complex and thereby in alterations in CCE. We will test our model by showing the existence of an endogenous complex and identifying protein-protein interactions responsible for complex assembly. Next, we will measure CCE in cells transfected with PKD1, PKD2 and TRPC1. We will evaluate an effect of PKD2 on CCE by introducing dominant negative constructs of PKD2 in cell lines that endogenously express PKD1, PKD2 and TRPC1 and testing whether wild type or mutant PKD2 can regulate cAMP accumulation in kidney epithelial cells. Our ultimate goal is to develop a biologically significant system that would allow us to probe the mechanisms by which pathogenic mutations in PKD2 alter its normal function, and to design therapeutic interventions in diseases such as ADPKD.

描述（由申请人提供）：绝大多数常染色体显性多囊肾病病例的基因功能 (ADPKD) 未知。根据序列分析，该基因产物多囊肾病基因 1 (PKD1) 被提议编码一种蛋白质在细胞-细胞和/或细胞-细胞外基质相互作用中发挥作用，同时 PKD2 被认为具有阳离子通道的功能。我们已经证明 PKD2 PKD2 在物理上与 PKD1 相关，并且只有在 PKD1 存在的情况下，PKD2 才能够形成Ca++可渗透的阳离子通道。除了 PKD 1 之外，还包括 PKD2 能够与瞬时受体电位通道 1 (TRPC1) 相关。我们现在表明 TRPC1 在上皮结构中广泛分布，主要是肾脏和肝脏的导管细胞。 TRPC 1 被证明可以增强 Ca++ 进入响应存储耗尽（或电容性 Ca++ 进入，CCE）。 CCE是非兴奋性细胞调节其功能的主要途径细胞内 Ca++ 浓度。在众多受调节的细胞功能中通过 CCE，cAMP 积累的调节是一种具有良好特征和特异性的机制。 CCE的生理目标。值得注意的是，cAMP 参与囊肿形成在肾和肝上皮细胞中的作用已得到很好的证实。我们建议 PKD1、PKD2 和 TRPC1 组装成功能复合物以增强 CCE。自然 PKD2 中发生的突变可能会导致该复合物的破坏从而导致 CCE 的改变。我们将通过证明存在性来测试我们的模型内源性复合物的形成和蛋白质-蛋白质相互作用的鉴定负责复杂的装配。接下来，我们将测量细胞中的 CCE 转染 PKD1、PKD2 和 TRPC1。我们将评估 PKD2 对 CCE 通过在细胞系中引入 PKD2 的显性失活构建体内源表达 PKD1、PKD2 和 TRPC1，并测试野生型或突变体 PKD2 可以调节肾上皮细胞中 cAMP 的积累。我们的最终目标是开发一个具有生物学意义的系统我们探究 PKD2 致病性突变改变其功能的机制正常功能，并设计针对疾病的治疗干预措施，例如 ADPKD。