Signaling Complexes in Renal Epithelial Cells

肾上皮细胞中的信号复合物

• 批准号: 6548642
• 负责人: Richard Tyler Miller
• 金额: $ 25.03万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6548642
• 关键词: G protein; JUN kinase; actin binding protein; biological signal transduction; calcium binding protein; calcium flux; caveolins; cell cell interaction; cell line; electron microscopy; enzyme induction /repression; epithelium; intracellular membranes; kidney cell; laboratory rat; mitogen activated protein kinase; parathyroid hormones; phospholipase C; protein localization; protein protein interaction; renal tubular transport; tissue /cell culture; transfection; yeast two hybrid system

DESCRIPTION (provided by applicant): The hypothesis guiding these studies is that the character and specificity of cell signaling systems is determined by structural proteins that organize multiple signaling proteins into discrete complexes in the cell. Some components are common to many signaling systems, while others may be unique. These studies will focus on the interaction of the extracellular Ca-sensing receptor (CaR) and filamin (ABP-280), a cytoskeletal protein, in renal epithelial cells with emphasis on MTAL and INCO cells. The CaR is a G protein-coupled receptor that responds to Ca and other polycations to regulate PTH secretion by the parathyroid glands and Ca, NaCI, KCl, and H20 transport in the kidney. The CaR has unique signaling properties that are not fully explained by its activation of Gi- and Gq-dependent signaling pathways. In an effort to identify potential scaffolding proteins for the signaling systems controlled by the CaR and identify new signaling pathways through which it might act, we used the CaR intracellular C-terminus as "bait" to screen a human kidney cDNA library using the yeast two hybrid approach. We found that the CaR interacts with filamin (ABP-280), a 280 kDa actin-binding cytoskeletal protein. Filamin also binds to variety of other signaling molecules including SEKI (MKK4), caveolin, the dopamine 02 receptor, Rho GTPases, SMADs and a- and Beta integrins, suggesting that it can act as a scaffold. The goals of this application are to define the interaction of the CaR and filamin and to understand how this interaction affects the signaling functions of the CaR in renal epithelial cells. The specific aims are: Aim 1) Characterize the interaction of the C-terminus of the CaR with fliamin using the yeast two hybrid system and mammalian cells; Aim 2) Determine the functional significance of the CaR-filamin interaction by analysis of CaR-stimulated signals in cells that lack filamin and by specific disruption of CaR-filamin association; Aim 3) Establish the subcellular pattern of distribution of the CaR in renal epithelial cells and determine if the interaction of the CaR with filamin contributes to the localization of the CaR in renal epithelial cells; Aim 4) Identify the smallest portion of filamin that can reconstitute signaling by the CaR in filamin-null cells, and then identify additional proteins that interact with that fragment. These studies will test the hypothesis that the interaction of filamin and the CaR is important for appropriate cell signaling by the CaR and that filamin acts as a scaffolding protein to organize the signaling pathway(s) regulated by the CaR.

描述(申请人提供)：指导这些研究的假设是，细胞信号系统的特征和特异性是由结构蛋白决定的，这些结构蛋白在细胞中将多个信号蛋白组织成离散的复合体。有些组件是许多信令系统通用的，而其他组件可能是独一无二的。这些研究将集中在细胞外钙敏感受体(CAR)和细丝蛋白(ABP-280)在肾上皮细胞中的相互作用，重点是MTAL和INCO细胞。CAR是一种G蛋白偶联受体，对钙和其他聚阳离子作出反应，以调节甲状旁腺分泌甲状旁腺激素和肾脏中钙、NaCI、KCl和H20的运输。汽车具有独特的信号特性，这不能完全用它激活依赖于GI和GQ的信号通路来解释。为了确定CAR调控的信号系统的潜在支架蛋白，并确定它可能通过的新的信号通路，我们以CAR细胞内的C-末端为“诱饵”，采用酵母双杂交的方法筛选了一个人肾cDNA文库。我们发现CAR与细丝蛋白(ABP-280)相互作用，丝氨酸是一种280 kDa的肌动蛋白结合的细胞骨架蛋白。丝素还可以与其他信号分子结合，包括Seki(MKK4)、小窝蛋白、多巴胺02受体、Rho GTP酶、Smads和β整合素，这表明它可以作为支架。这项应用的目标是定义CAR和细丝蛋白的相互作用，并了解这种相互作用如何影响CAR在肾上皮细胞中的信号功能。其具体目的是：1)利用酵母双杂交系统和哺乳动物细胞鉴定CAR的C末端与Fliamin的相互作用；2)通过分析缺少细丝的细胞中的CAR刺激信号以及通过特异性地破坏CAR与细丝的结合来确定CAR-细丝相互作用的功能意义；目的3)建立CAR在肾上皮细胞中的亚细胞分布模式，并确定CAR与细丝的相互作用是否有助于CAR在肾上皮细胞中的定位；目的4)确定在细丝蛋白缺失细胞中可以通过CAR重组信号的最小丝蛋白部分，然后确定与该片段相互作用的其他蛋白质。这些研究将检验这样一种假设，即细丝蛋白和CAR的相互作用对于CAR适当的细胞信号传递非常重要，并且细丝蛋白充当支架蛋白来组织由CAR调控的信号通路(S)。