Signaling Complexes in Renal Epithelial Cells

肾上皮细胞中的信号复合物

基本信息

批准号：
6640447
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
项目类别：
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

项目摘要

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的，结构蛋白将多个信号蛋白组织成细胞中离散的复合物。一些组件对于许多信号系统来说是通用的，而另一些组件可能是独特的。这些研究将重点关注肾上皮细胞中细胞外 Ca 感应受体 (CaR) 和细丝蛋白 (ABP-280)（一种细胞骨架蛋白）的相互作用，重点是 MTAL 和 INCO 细胞。 CaR 是一种 G 蛋白偶联受体，可响应 Ca 和其他聚阳离子来调节甲状旁腺的 PTH 分泌以及肾脏中的 Ca、NaCI、KCl 和 H20 转运。 CaR 具有独特的信号传导特性，但其激活 Gi 和 Gq 依赖性信号传导途径并不能完全解释这些特性。为了鉴定由 CaR 控制的信号系统的潜在支架蛋白，并确定其可能发挥作用的新信号通路，我们使用 CaR 细胞内 C 末端作为“诱饵”，使用酵母两种方法筛选人肾 cDNA 文库。混合方法。我们发现 CaR 与细丝蛋白 (ABP-280) 相互作用，细丝蛋白是一种 280 kDa 肌动蛋白结合细胞骨架蛋白。细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、SMAD 以及 a- 和 Beta 整联蛋白，表明它可以充当支架。该应用的目标是定义 CaR 和细丝蛋白的相互作用，并了解这种相互作用如何影响肾上皮细胞中 CaR 的信号传导功能。具体目标是：目的 1) 使用酵母二杂交系统和哺乳动物细胞表征 CaR C 末端与 fliamin 的相互作用；目标 2) 通过分析缺乏细丝蛋白的细胞中 CaR 刺激的信号以及特异性破坏 CaR-细丝蛋白关联，确定 CaR-细丝蛋白相互作用的功能意义；目的3)建立肾上皮细胞中CaR的亚细胞分布模式，并确定CaR与细丝蛋白的相互作用是否有助于CaR在肾上皮细胞中的定位；目标 4) 鉴定细丝蛋白缺失细胞中可以通过 CaR 重建信号传导的最小部分，然后鉴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：细丝蛋白和 CaR 的相互作用对于 CaR 的适当细胞信号传导非常重要，并且细丝蛋白充当支架蛋白来组织由 CaR 调节的信号通路。