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

描述（由申请人提供）：负责基因的功能 对于绝大多数常染色体显性遗传性多囊肾病病例， （ADPKD）未知。基于序列分析， 已提出多囊肾病基因1（PKD 1）编码蛋白质 在细胞-细胞和/或细胞-细胞外基质相互作用中起作用， PKD 2被认为是阳离子通道。我们已经证明PKD 2 与PKD 1有身体上的联系，只有在PKD 1存在的情况下，PKD 2才能 以形成Ca++可渗透的阳离子通道。除PKD 1外，PKD 2也是 能够与瞬时受体电位通道1（TRPC 1）结合。我们 现在显示TRPC 1在上皮结构中具有广泛的分布， 主要是肾脏和肝脏的导管细胞。TRPC 1被证明可以增强 Ca++内流对钙池耗竭的响应（或容量性Ca++内流，CCE）。 CCE是非兴奋性细胞调节其细胞周期的主要途径。 细胞内Ca++浓度。在调节的许多细胞功能中， 通过CCE，cAMP积累的调节是一个充分表征的和特异性的， CCE的生理指标。值得注意的是，cAMP参与囊肿形成， 在肾脏和肝脏上皮细胞中的作用已经很好地确立。我们建议 PKD 1、PKD 2和TRPC 1组装成功能复合物以增强CCE。自然 PKD 2中发生的突变可能导致这种复合物的破坏， 从而改变CCE。我们将通过证明存在 和鉴定蛋白质间的相互作用 负责复杂的装配。接下来，我们将测量细胞中的CCE 用PKD 1、PKD 2和TRPC 1转染。我们将评估PKD 2对 CCE通过在细胞系中引入PKD 2的显性阴性构建体， 内源性表达PKD 1、PKD 2和TRPC 1，并测试野生型或 突变PKD 2可调节肾上皮细胞中cAMP的积累。我们 最终目标是开发一个具有生物学意义的系统， 我们探索PKD 2致病突变改变其致病性的机制， 正常功能，并设计疾病的治疗干预措施， 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.