Signaling Complexes in Renal Epithelial Cells

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的，结构蛋白将多种信号蛋白组织成细胞内离散的复合物。有些组件是许多信号系统共同的，而其他组件可能是独特的。这些研究将集中于细胞外钙感应受体（CaR）和纤维蛋白（ABP-280）（一种细胞骨架蛋白）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，响应Ca和其他聚合，调节甲状旁腺的甲状旁腺分泌PTH以及Ca、NaCI、KCl和H20在肾脏中的转运。CaR具有独特的信号特性，不能完全解释其激活的Gi和gq依赖的信号通路。为了鉴定由CaR控制的信号系统的潜在支架蛋白，并鉴定它可能通过的新的信号通路，我们使用CaR细胞内c端作为“诱饵”，使用酵母双杂交方法筛选人类肾脏cDNA文库。我们发现CaR与丝蛋白（ABP-280）相互作用，这是一种280 kDa的肌动蛋白结合细胞骨架蛋白。丝蛋白还与多种其他信号分子结合，包括SEKI （MKK4）、caveolin、多巴胺02受体、Rho GTPases、SMADs以及a-和β整合素，这表明它可以作为支架。本应用程序的目的是定义CaR和丝蛋白的相互作用，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。具体目的是：目的1)利用酵母双杂交系统和哺乳动物细胞表征CaR的c端与fliamin的相互作用；目的2)通过分析缺乏丝蛋白的细胞中的car刺激信号和特异性破坏car -丝蛋白结合来确定car -丝蛋白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与丝蛋白的相互作用是否有助于CaR在肾上皮细胞中的定位；目的4)在无丝蛋白的细胞中，鉴定出可以重建CaR信号的最小部分丝蛋白，然后鉴定出与该片段相互作用的其他蛋白质。这些研究将验证以下假设，即丝蛋白和CaR的相互作用对于CaR的适当细胞信号传导很重要，并且丝蛋白作为支架蛋白组织由CaR调节的信号传导途径。

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.