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

描述(申请人提供)：导致绝大多数常染色体显性遗传性多囊肾病病例的基因的功能(S) (ADPKD)是未知的。根据序列分析，该基因产物 多囊肾病基因1(PKD1)被认为是一种编码蛋白质 在细胞-细胞和/或细胞-细胞外基质相互作用中发挥作用 PKD2被认为是一种阳离子通道。我们已经证明了PKD2 PKD2在物理上与PKD1相关，并且只有在PKD1存在时，PKD2才能 形成钙离子渗透性阳离子通道。除了PKD 1，PKD2也是 能够与瞬时受体电位通道1(TRPC1)相联系。我们 现在显示TRPC1在上皮结构中有广泛的分布， 主要是肾脏和肝脏的导管细胞。TRPC 1被证明增强了 响应于存储器耗尽的钙离子内流(或容量钙离子内流，CCE)。 CCE是非兴奋性细胞调节其功能的主要途径。 细胞内钙离子浓度。在众多受调控的细胞功能中 CCE对cAMP积累的调控具有很好的特征性和特异性 CCE的生理指标。值得注意的是，cAMP参与了包囊的形成 在肾脏和肝脏上皮细胞已经建立得很好。我们建议 PKD1、PKD2和TRPC1组装成一个功能复合体，增强CCE。自然而然 PKD2发生突变可能导致该复合体的破坏和 从而在CCE中发生变化。我们将通过展示模型的存在性来测试我们的模型 内源性复合体和识别蛋白质-蛋白质相互作用 负责复杂的装配工作。接下来，我们将以细胞为单位测量CCE 分别用PKD1、PKD2和TRPC1基因转染。我们将评估PKD2对 CCE通过在细胞系中引入PKD2的显性负性结构来实现 内源性表达PKD1、PKD2和TRPC1并检测野生型或 突变型PKD2可调节肾上皮细胞内cAMP的积聚。我们的 最终目标是开发一种具有生物学意义的系统，它将允许 美国将探讨PKD2致病突变改变其功能的机制 正常功能，并为疾病设计治疗干预措施，如 ADPKD.

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.