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的AT2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 在个体发育和组织通过细胞凋亡重塑过程中的数量。异常 在AT中，受体功能参与了病理的发展 情况如CAKUT(先天性肾脏和尿路异常)， 有缺陷的血管个体发生，梗死后异常的心脏重塑和 血管损伤后新生内膜的形成。其表达式被限制为 在成人体内有少量组织，但在胎儿发育过程中，组织 修复和重塑AT2受体在以下部位过度表达 改建。AT2受体是一种新的研究范式，因为它是一个7TM 具有调节MAPKs、caspase和蛋白质的独特能力的受体 G蛋白以外的磷酸酶。对……基础的全面理解 AT2受体的抗生长功能需要分析 结构-功能与信号分子的鉴定 这种受体的信号转导。进行详细的研究 涉及的机制，并确定介导细胞凋亡的分子 来自AT2受体的信号，我们已经建立了体外细胞模型 细胞凋亡。 本申请的具体目的是：(1)定义 AT2受体在启动细胞凋亡中的结构与功能关系。 (2)明确一种新的接头分子在AT2受体介导的p38中的作用 MAPK活化与细胞凋亡。(3)分离新的信号分子， 与AT受体相互作用，并确定它们在其功能中的作用。 在AT、受体诱导的细胞凋亡中有缺陷的定点突变体将 检查它们激活不同细胞内信号的能力 合作安装程序性细胞死亡。多种MAPK的激活 异构体、半胱氨酸酶、蛋白酪氨酸磷酸酶异构体、G-蛋白G介导型 磷酸酶；CDKs和CDK抑制剂的下调将是 在转基因的血管平滑肌细胞中进行了研究。这些研究将 描绘了CD环在耦合到不同区域中的协同作用 信号以及在定义受体诱导的AT所涉及的步骤中 死亡之路。我们将利用酵母双杂交技术进行互作克隆 AT2受体-CD！建造成一个诱饵。将识别出真正的阳性克隆 通过严格的测试，包括基因筛选、蛋白质相互作用研究和 功能互补分析。全长cdna将被表征为 它们在介导AT、受体诱导的细胞凋亡和表达变化中的作用 处于不同的病理生理状态。我们将使用突变，蛋白质 相互作用分析和共转染功能分析评价 AT2介导的p38MAPK激活中候选接头蛋白的特异性。 我们预计，这些研究将阐明调控机制。 AT2受体诱导血管内皮细胞和其他类型血管内皮细胞的凋亡 细胞。此外，这些研究可能会增加对 调节AT2受体介导的其他功能的机制，如 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.