POLYCYSTIN-1 MEDIATED CALCIUM AND cAMP SIGNALING

Polycystin-1 介导的钙和 Camp 信号传导

• 批准号: 7070152
• 负责人: JAMES P CALVET
• 金额: $ 20.21万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7070152
• 关键词: G protein; biological signal transduction; calcium flux; cell growth regulation; cell proliferation; cyclic AMP; gene expression; gene mutation; genetic manipulation; genetically modified animals; laboratory mouse; membrane proteins; molecular pathology; pathologic process; phenotype; polycystic kidney; protein protein interaction; protein structure function; receptor coupling; tissue /cell culture

Several observations have suggested that cAMP is central to the pathogenesis of PKD, by activating the Ras/MAPK pathway resulting in the cell proliferation required for cyst formation and cyst growth, and by activating CFTR to stimulate fluid secretion to fill cysts. The importance of cAMP was recently further confirmed by the successful treatment of PKD in four animal models, using a vasopressin V2 receptor antagonist which presumably lowered renal cAMP. However, while these experiments support the view that cAMP is important, it is not known how a defect in the polycystins causes misregulation of cAMP-mediated mechanisms associated with increased cell proliferation and cyst enlargement. The polycystins are thought to regulate intracellular Ca2+ levels in response to ligand-mediated or mechanosensory stimuli. Recently, we showed that polycystin-1 alone is capable of elevating intracellular Ca2+ through a heterotrimeric G protein-coupled mechanism. We have also shown, using three cell culture model systems, that PKD-like cell proliferation is dependent on cAMP-mediated activation of the Ras/MAPK pathway and to changes in Ca2+-dependent gene expression. All three systems displayed the same phenotypic switch from cAMP-inhibited to cAMP-stimulated cell proliferation. Yet, despite these observations, it is still not clear whether and how the polycystins may be involved. As such, we plan to determine if the PKD-like cAMP-stimulated phenotype is a characteristic of Pkd1 deficient cells. We will use the Pkd1(m1Bei) mouse, which has a single amino acid missense mutation and a full-blown PKD phenotype, and we plan to generate two new mouse models, Pkd1(DeltaL) and Pkd1(Gpro), which will carry, respectively, a single amino acid (aa) deletion mutation in the heterotrimeric G protein binding region of polycystin-1 mimicking a known human mutation, or a 52 aa frameshift mutation in the heterotrimeric G protein binding region of polycystin-1. We also plan to utilize metanephric kidney cultures from these mice to determine whether the PKD-like phenotypic switch occurs in embryonic kidney cells at a time when cysts begin to grow in Pkd1-deficient mouse models and in human ADPKD. Finally, we plan to test polycystin-1 mediated signaling activity under conditions of fluid flow mediated ciliary bending to determine if polycystin-1 can generate a Ca2+ signal sufficient to alter Ca2+- dependent gene expression.

一些观察表明，cAMP 通过激活 Ras/MAPK 途径导致囊肿形成和囊肿生长所需的细胞增殖，并通过激活 CFTR 刺激液体分泌以填充囊肿，是 PKD 发病机制的核心。最近，在四种动物模型中使用加压素 V2 受体拮抗剂成功治疗 PKD，进一步证实了 cAMP 的重要性，该拮抗剂可能会降低肾脏 cAMP。然而，虽然这些实验支持cAMP很重要的观点，但尚不清楚多囊蛋白的缺陷如何导致与细胞增殖增加和囊肿扩大相关的cAMP介导机制的失调。多囊蛋白被认为可响应配体介导或机械感觉刺激来调节细胞内 Ca2+ 水平。最近，我们 研究表明，单独的多囊蛋白-1 能够通过异源三聚体 G 蛋白偶联机制升高细胞内 Ca2+。我们还使用三种细胞培养模型系统证明，PKD 样细胞增殖依赖于 cAMP 介导的 Ras/MAPK 通路激活以及 Ca2+ 依赖性基因表达的变化。所有三个系统都显示出相同的表型转换，即从 cAMP 抑制到 cAMP 刺激的细胞增殖。然而，尽管有这些观察结果，仍不清楚多囊蛋白是否以及如何参与其中。因此，我们计划确定 PKD 样 cAMP 刺激表型是否是一种 Pkd1 缺陷细胞的特征。我们将使用 Pkd1(m1Bei) 小鼠，它具有单个氨基酸错义突变和成熟的 PKD 表型，我们计划生成两种新的小鼠模型，Pkd1(DeltaL) 和 Pkd1(Gpro)，它们将携带，分别是，多囊蛋白-1 的异源三聚体 G 蛋白结合区中的单个氨基酸 (aa) 缺失突变模仿了已知的人类突变，或多囊蛋白-1 中的 52 个氨基酸移码突变。多囊蛋白-1 的异三聚体 G 蛋白结合区。我们还计划利用这些小鼠的后肾肾培养物来确定当 Pkd1 缺陷小鼠模型和人类 ADPKD 中囊肿开始生长时，胚胎肾细胞中是否会发生 PKD 样表型转换。最后，我们计划在流体流动介导的纤毛弯曲条件下测试多囊蛋白-1介导的信号活动，以确定多囊蛋白-1是否可以产生足以改变Ca2+-的Ca2+信号。 依赖性基因表达。