G PROTEIN-DEPENDENT SIGNALING IN RENAL EPITHELIAL CELLS

这项研究计划的长期目标是了解 G蛋白依赖的细胞内分子机制 信号系统调节肾上皮细胞的功能。G蛋白- 偶联受体(如对于细胞外钙、A11和AVP)激活 多条平行通路涉及不同的G蛋白、激酶、 和转录因子，以提供集成的控制网络 调节多种细胞功能。尽管事实是这些配体 这些受体及其对第二信使和 已经对运输进行了相当详细的研究，其机制 信息在细胞中传播的方式仍然不完全 定义是因为：1)许多蛋白质在 结构和功能；2)低水平表达；3) 化学激活剂或抑制剂在许多已知情况下并不存在 蛋白质；以及4)许多蛋白质可能仍有待鉴定。 本课程将研究细胞外钙受体(CaO-R)所传递的信号 使用允许分离信令过程的模型系统 转换为可精确分析的组件，以测试特定的 假设。其组成如下：1)受体-G的模式 蛋白质偶联；2)个体激活细胞内的激酶 G蛋白；3)Na/H交换启动子(NHE-1)的激活 通过单独的G蛋白和激酶；以及4)鉴定 NHE-1启动子元件对G蛋白和 激活剂。我们要检验的假设是：1)钙氧受体激活 多个G蛋白(Gq~G12‘和一个Gi)：2) 通过不同的机制耦合到CaO-R信号，并激活 不同组的MAP激酶；3)G蛋白和激酶激活 NHE-1启动子的不同元件；以及4)信号 调节Na/H交换也调节体内其他转运蛋白 协调一致的方式。研究将在细胞中进行， 允许同时进行遗传和生化分析，新鲜的 分离MTAL细胞以验证遗传和生化研究 并培养MTAL细胞，以测试这些信号的影响 关于运输监管的制度。通过以下方式建立机制 CaO-R的作用将提高我们对基因的理解 与CaO-R和电解质异常有关的疾病和 间质性肾炎伴发高钙血症。

The long term goals of this research program is to understand the intracellular molecular mechanisms by which G protein-dependent signaling systems regulate renal epithelial cell function. G protein- coupled receptors (eg. for extracellular Ca, A11, and AVP) activate multiple parallel pathways involving distinct G proteins, kinases, and transcription factors to provide an integrated control network to regulate multiple cell functions. Despite the fact that the ligands for these receptors and their effects on second messengers and transport have been studied in considerable detail, the mechanisms by which information travels through the cell remain incompletely defined because: 1) many of the proteins are closely related in structure and function; 2) they are expressed at low levels; 3) chemica activators or inhibitors do not exist for many known proteins; and 4) many proteins probably remain to be identified. Signaling by the extracellular Ca receptor (Cao-R) will be examined using a model system that allows separation of the signaling process into components that can be analyzed with precision to test specific hypotheses. The components are: 1) the pattern of receptor - G protein coupling; 2) activation of intracellular kinases by individual G proteins; 3) activation of the Na/H exchanger promoter (NHE-1) by individual G proteins and kinases; and 4) identification of the NHE-1 promoter elements that respond to the G proteins and kinases. The hypotheses we will test are: 1) the Ca o -R activates of multiple G proteins (Gq~ G12' and a Gi): 2) the G proteins that couple to the Cao-R signal by different mechanisms, and activate different sets of MAP kinases; 3) the G proteins and kinase activate different elements of the NHE-1 promoter; and 4) the signals that regulate Na/H exchange also regulate other transporters in a coordinated manner. The studies will be performed in cells that allow simultaneous genetic and biochemical analyses, freshly isolated MTAL cells to verify the genetic and biochemical studies and cultured MTAL cells to test the effects of these signaling systems on regulation of transport. Establishing the mechanisms by which the Cao-R acts will improve our understanding of the genetic diseases linked to the Cao-R and the electrolyte abnormalities and interstitial nephritis associated with hypercalcemia.