Angiotensin II Receptor Signaling Domains

描述（由申请人提供）：血管紧张素II（Ang II）是一种有效的血管肥大刺激物，其通过血管平滑肌细胞（VSMC）中的Ang II 1型受体（AT 1 R）介导。它的许多重要输出来自EGF受体（EGF-R）的反式激活，其需要cSrc的初始激活并且依赖于来自NAD（P）H氧化酶（Nox）的活性氧物质（ROS）。小窝蛋白富集脂筏（CE/LRM）是一种特殊的膜微结构域，其中信号分子如EGFR通过与小窝蛋白-1（Cav 1）相互作用而被区室化。我们发现Ang II促进AT 1 R向CE/LRM的运输，这是EGF-R反式激活和EGF-R从CE/LRM流出所必需的，导致pY-EGFR与pY 14 Cav 1在粘着灶共定位。我们还发现，AT 1 R迁移到CE/LRM是依赖于ROS，cSrc，微管/肌动蛋白细胞骨架和Cav 1;然而，基本的，组织的分子机制知之甚少。cAbl是连接受体酪氨酸激酶和肌动蛋白重塑的F-肌动蛋白结合非受体酪氨酸激酶。Cavl是cAbl的主要ROS依赖性酪氨酸磷酸化靶标。我们发现，AT 1 R激活VSMCs和小鼠主动脉中的cAbl，并促进cAbl与AT 1 R的结合，这依赖于ROS，cSrc和Cav 1。初步数据使我们假设cAbl作为ROS、Cav 1和肌动蛋白细胞因子依赖性AT 1 R运输和信号传导的中心组织者发挥作用，这可能有助于血管肥大。为了验证这一假设，AIM 1将表征AT 1 R和cAbl的相互作用，并定义Ang II激活cAbl的机制。AIM 2将探索cAbl被Nox衍生的ROS激活的机制，重点是SHP-2的氧化失活，SHP-2直接与AT 1 R结合并负调节cSrc。本研究将探讨cAbl介导Ang II刺激的AT 1 R迁移到CE/LRM、在局灶性粘连和肥大处形成pY-EGF-R信号复合物的分子机制，重点关注cAbl下游靶点cornein和Y14 Cav 1。AIM 4将评估cAbl在体内Ang II诱导的血管肥大中的功能作用。将使用敲除小鼠和细胞、活细胞成像、细胞分级分离、蛋白质-蛋白质相互作用和分子生物学分析。这些研究将为血管紧张素II信号传导的组织提供新的见解，并为心血管疾病的新治疗方法确定潜在的新靶点。

DESCRIPTION (provided by applicant): Angiotensin II (Ang II) is a potent stimulator of vascular hypertrophy which is mediated through the Ang II type l receptor (AT1R) in vascular smooth muscle cells (VSMCs). Many of its important outputs result from transactivation of the EGF receptor (EGF-R), which requires initial activation of cSrc and which is dependent on reactive oxygen species (ROS) derived from NAD(P)H oxidase (Nox). Caveolin-enriched lipid rafts (CE/LRM) are specialized membrane microdomains where signaling molecules such as EGFR are compartmentalized via interacting with caveolin-1 (Cav1). We showed that Ang II promotes AT 1R trafficking into CE/LRM, which is required for transactivation of EGF-R and egress of EGF-R from the CE/LRM, resulting in colocalization of pY-EGFR with pY14Cav1 at focal adhesions. We also found that AT1R migration into CE/LRM is dependent on ROS, cSrc, microtubules/actin cytoskeleton and Cav1; however, underlying, organizing molecular mechanisms are poorly understood. cAbl is a F-actin binding non-receptor tyrosine kinase that links receptor tyrosine kinase and actin remodeling. Cav1 is a major ROS-dependent tyrosine phosphorylation target of cAbl. We show that AT1R activates cAbl in VSMCs and mouse aorta, and promotes cAbl binding to the AT1R, which are dependent on ROS, cSrc and Cav1. Preliminary data led us to hypothesize that cAbl functions as a central organizer for ROS, Cav1 and actin cytoskeleton-dependent AT1R trafficking and signaling, which may contribute to vascular hypertrophy. To test this hypothesis, AIM1 will characterize the interaction of AT1R and cAbl, and define the mechanisms of cAbl activation by Ang II. AIM2 will explore the mechanisms by which cAbl is activated by Nox-derived ROS with focusing on oxidative inactivation of SHP-2 which binds directly to AT1R and negatively regulates cSrc. AIMS will explore the molecular mechanisms by which cAbl mediates Ang ll-stimulated AT1R migration into CE/LRM, formation of pY-EGF-R signaling complex at focal adhesions and hypertrophy with focus on cAbl downstream targets, cortactin and Y14Cav1. AIM4 will assess the functional role of cAbl in Ang ll-induced vascular hypertrophy in vivo. Knockout mice and cells, live cell imaging, cell fractionation, protein-protein interaction and molecular biological analysis will be used. These studies should provide new insights into the organization of Ang II signaling and identify potential new targets for novel therapeutic approaches to cardiovascular diseases.