Angiotensin II Receptor Signaling Domains

描述(申请人提供)：血管紧张素II(Ang II)是一种强大的血管肥厚刺激因子，它通过血管平滑肌细胞(VSMC)上的Ang II型L受体(AT1R)介导。它的许多重要输出是EGF受体(EGF-R)反式激活的结果，这需要中国证监会的初始激活，并依赖于NAD(P)H氧化酶(NOx)产生的活性氧物种(ROS)。富含小窝蛋白的脂筏(CE/LRM)是一类特殊的膜微区，EGFR等信号分子通过与小窝蛋白-1(Caveolin-1，Cav1)相互作用而被分割。我们发现Ang II促进AT1R转运到CE/LRM，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pY14Cav1在局部粘连处共存。我们还发现AT1R向CE/LRM的迁移依赖于ROS、CSRC、微管/肌动蛋白细胞骨架和Cav1；然而，其潜在的组织分子机制尚不清楚。CABL是一种F-肌动蛋白结合的非受体酪氨酸激酶，连接受体酪氨酸激酶和肌动蛋白重塑。Cav1是CABL主要依赖ROS的酪氨酸磷酸化靶标。我们发现AT1R激活VSMCs和小鼠主动脉中的CABL，并促进CABL与AT1R的结合，这依赖于ROS、CSRC和Cav1。初步数据使我们假设CABL作为ROS、Cav1和肌动蛋白依赖的AT1R运输和信号的中心组织者，这可能有助于血管肥厚。为了验证这一假说，AIM1将描述AT1R和CABL的相互作用，并确定Ang II激活CABL的机制。AIM2将探索NOx来源的ROS激活CABL的机制，重点是直接与AT1R结合的SHP-2的氧化失活，并负向调节CSRC。AIMS将探索CABL介导AngⅡ刺激的AT1R迁移到CE/LRM的分子机制，在局部粘连和肥大处形成Py-EGF-R信号复合体，重点关注CABL下游靶点Cortactin和Y14Cav1。AIM4将评估CABL在体内AngⅡ诱导的血管肥大中的作用。将使用基因敲除小鼠和细胞、活细胞成像、细胞分离、蛋白质-蛋白质相互作用和分子生物学分析。这些研究应该为Ang II信号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.