Signaling Complexes in Renal Epithelial Cells

肾上皮细胞中的信号复合物

• 批准号: 6761743
• 负责人: Richard Tyler Miller
• 金额: $ 21.93万
• 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 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6761743
• 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（由申请人提供）：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将多种信号蛋白组织到细胞中离散复合物中的结构蛋白。有些组件对于许多信号系统都是共同的，而另一些组件可能是唯一的。这些研究将集中于肾上皮细胞中细胞骨骼蛋白（ABP-280）的相互作用，重点是MTAL和INCO细胞。该汽车是G蛋白偶联受体，对CA和其他多阳离子有反应，以调节甲状旁腺和CA，NACI，KCL和H20在肾脏中的PTH分泌。该汽车具有独特的信号传导特性，其激活GI-和GQ依赖性信号通路并未完全解释。为了确定由汽车控制的信号系统的潜在脚手架蛋白并确定其可能作用的新信号通路，我们使用CAR内C-末端作为“诱饵”来筛选人类肾脏cDNA库，并使用酵母菌两混合方法。我们发现该汽车与280 kDa肌动蛋白结合细胞骨架蛋白的Filamin（ABP-280）相互作用。丝蛋白还与包括SEKI（MKK4），小窝蛋白，多巴胺02受体，Rho GTPases，Smads，Smads以及A-和β整合素在内的其他信号分子结合，表明它可以充当脚手架。该应用的目标是定义汽车和丝蛋白的相互作用，并了解这种相互作用如何影响肾上皮细胞中汽车的信号传导功能。具体目的是：目标1）表征使用酵母两个杂种系统和哺乳动物细胞的汽车C末端与氟氨酸的相互作用；目标2）通过分析缺乏丝蛋白的细胞中的CAR刺激信号和CAR-Filamin关联的特异性破坏来确定CAR-FILAMIN相互作用的功能显着性；目标3）建立在肾上皮细胞中CAR分布的亚细胞分布模式，并确定汽车与丝蛋白的相互作用是否有助于汽车在肾上皮细胞中的定位；目标4）确定最小的丝蛋白可以在丝蛋白无细胞中通过汽车重建信号传导，然后确定与该片段相互作用的其他蛋白质。这些研究将检验以下假设：丝蛋白和汽车的相互作用对于汽车的适当细胞信号很重要，并且丝蛋白充当脚手架蛋白，以组织由汽车调节的信号通路。