Mechanism of Cell Death in Expressing AT2 Receptor

描述（由申请人提供）：我们广泛的长期目标是 了解血管紧张素II（Ang）AT 2受体的抗生长功能 II）。越来越多的证据表明，AT.，受体调节细胞 在个体发育和通过细胞凋亡重塑组织过程中的数量。异常 在AT中，受体功能与病理性 诸如CAKUT（先天性肾和泌尿道异常）的病症， 血管的个体发育缺陷，梗死后心脏重塑异常， 血管损伤后新生内膜形成。其表达仅限于 成年人有少量的组织，但在胎儿发育期间， 修复和重塑AT 2受体在以下部位过度表达： 重塑AT 2受体是一种新的研究范式，因为它是一种7 TM受体。 具有调节MAPKS、半胱天冬酶和蛋白质的独特能力的受体 除了G蛋白。全面理解的基础 AT 2受体的抗生长功能需要分析 结构-功能和信号分子的鉴定 从这个受体的传导。进行详细研究， 参与的机制，并确定介导凋亡的分子 从AT 2受体的信号，我们已经建立了体外细胞模型， 凋亡 本申请的具体目的是：（1）定义 AT 2受体在启动细胞凋亡中的结构-功能关系。 (2)为了确定一种新的衔接分子在AT 2受体介导的p38中的作用， MAPK活化和凋亡。(3)分离出新的信号分子， 与AT受体相互作用，并表征其在其功能中的作用。 AT的定点突变缺陷，受体诱导的细胞凋亡将被抑制。 检测它们激活不同细胞内信号的能力， 协同地引起程序性细胞死亡。各种MAPK的激活 亚型，半胱天冬酶，蛋白酪氨酸磷酸酶亚型，G蛋白G介导 磷酸酶;和下调的Cdks和Cdk抑制剂将是 在转染的血管平滑肌细胞中研究。这些研究将 描绘了CD环在与不同的 信号和定义参与AT的步骤，受体诱导的 死亡之路我们将利用酵母双杂交技术进行酵母双杂交相互作用克隆， AT 2受体CD！作为诱饵。将鉴定出真实的阳性克隆 通过严格的测试，包括基因筛选，蛋白质相互作用研究， 功能互补分析将对全长cDNA进行表征， 它们在介导AT、受体诱导的凋亡和表达变化中的作用 各种病理生理状态。我们将使用诱变，蛋白质 通过共转染进行相互作用测定和功能分析，以评估 候选衔接蛋白在AT 2介导的p38 MAPK激活中的特异性。 我们预计这些研究将阐明调节 AT 2受体在平滑肌和其他类型的平滑肌细胞中诱导细胞凋亡， 细胞此外，这些研究可能会增加对 调节其他AT 2受体介导的功能的机制， 心脏保护和胎儿发育。

DESCRIPTION (provided by the applicant): Our broad, long term objective is to understand the antigrowth functions of the AT2 receptor for angiotensin II (Ang II). Accumulating evidences suggests that the AT., receptor regulates cell number during ontogenesis and remodeling of tissues by apoptosis. Abnormality in AT, receptor function is implicated in development of pathological conditions such as CAKUT (congenital abnormality of kidney and urinary tract), flawed ontogenesis of vessels, abnormal post-infarct cardiac remodeling and in neointima formation following vascular injury. Its expression is restricted to a small number of tissues in adults, but during fetal development, tissue repair and remodeling the AT2 receptor is over expressed at sites of remodeling. The AT2 receptor is a novel paradigm for study since it is a 7TM receptor with the unique ability to regulate MAPKS, caspases and protein phosphatases besides G proteins. A complete understanding of the basis of antigrowth functions of the AT2 receptor requires the analysis of structure-function and the identification of molecules involved in signal transduction from this receptor. To undertake a detailed study of the mechanisms involved and to identify the molecules that mediate apoptotic signals from the AT2 receptor, we have established in vitro cell models of apoptosis. The specific aims for this application are: (1) To define the structure-function relationship of the AT2 receptor in initiating apoptosis. (2) To define the role of a novel adaptor molecule in AT2 receptor-mediated p38 MAPK activation and apoptosis. (3) To isolate novel signaling molecules that interact with the AT, receptor and to characterize their role in its functions. Site-directed mutants defective in AT, receptor-induced apoptosis will be examined for their ability to activate different intracellular signals that co-operatively mount programmed cell death. The activation of various MAPK isoforms, caspases, protein tyrosine phosphatase isoforms, G-protein G-mediated phosphatases; and the down regulation of Cdks and Cdk-inhibitors will be studied in transfected vascular smooth muscle cells. These studies will delineate the cooperative interaction of CD-loops in coupling to distinct signals and in defining the steps that are involved in the AT, receptor-induced death pathway. We will carry out yeast two-hybrid interaction cloning using the AT2 receptor-CD! construct as a bait. Real positive clones will be identified by rigorous tests including genetic screening, protein interaction studies and functional complementation analysis. Full length cDNA will be characterized for their role in mediating AT, receptor-induced apoptosis and expression changes in various pathophysiological states. We will use mutagenesis, protein interaction assays and functional analysis by co-transfection to evaluate the specificity of a candidate adapter protein in AT2-mediated p38 MAPK activation. We anticipate that these studies will shed light on the mechanisms regulating induction of apoptosis by the AT2 receptor in smooth muscle and other types of cells. Furthermore, these studies may lead to an increased understanding of the mechanisms regulating other AT2 receptor-mediated functions such as cardio-protection and fetal development.