Molecular Basis of Ang II Receptor Functions

说明(申请人提供)：血管紧张素受体阻滞剂(ARB)用于治疗高血压、心力衰竭和其他心血管疾病，可阻止血管紧张素1型受体(AT1)对血管紧张素II(Ang II)的激活和信号转导。Ang II与AT1受体结合可激活信号转导，从而导致细胞内钙离子的动员、蛋白激酶的激活和基因转录的激活。然而，Ang II或ARB通过与AT1受体结合来调控这些过程的分子机制尚不清楚。我们的长期目标是了解与血管紧张素Ⅱ结合的AT1受体如何激活多种信号转导过程，以及ARB如何影响这一过程。为了实现这一目标，我们开发了Ang II类似物和受体突变体，它们可以在没有G蛋白激活的情况下激活ERK信号。目前的提案将解决两个问题：(1)AT1选择性配体如何被引导到受体的结合口袋？以及(2)G蛋白介导的AT1受体信号与非G蛋白介导的AT1受体信号之间的差异在体内的后果是什么？我们将确定细胞外环(ECL)是否直接参与配体受体的相互作用以及AT1受体的激活/抑制。我们预计，ECL-2在受体的非活性状态(R)与TM结构域相互作用，在激活状态(特定目标1)与Ang II相互作用。我们发现在Ang II激活的细胞中，AT1受体与β-arrestin及其片段结合，这在其他条件下是不存在的。我们将检验这一假设，即特定部位的β-arrestin蛋白分解是一种具有功能后果的新信号模式。我们将确定在Ang II激活的细胞中β-arrestin的裂解位置及其功能后果(特定目标2)。G蛋白依赖的AT1受体信号不同于G蛋白非依赖性信号，因为它引起ERK的核转位。我们将确定血管紧张素Ⅱ/AT1受体诱导的ERK1/2核转位在基因表达激活中的作用(特定目标3)。这些研究对于更好地了解Ang II/AT1受体的作用机制和开发针对AT1受体的改进药物是必不可少的。

DESCRIPTION (provided by applicant): Angiotensin receptor blockers (ARB) used in the treatment of hypertension, heart-failure and other cardiovascular disease conditions prevents activation and signal transduction by the angiotensin type 1 receptor (AT1) for angiotensin II (Ang II). Binding of Ang II to the AT1 receptor activates signal-transduction that leads to mobilization of intracellular calcium, activation of protein kinases, and activation of gene transcription. Yet the molecular mechanism of modulation of these processes by Ang II or ARBs via binding to the AT1 receptor is not clear. Our long-term goal is to understand how the Ang ll-bound AT1 receptor activates the multiple signal transduction process and how ARBs affect this process. Towards this goal, we have developed Ang II- analogs and receptor mutants that activate the ERK-signaling in absence of the G-protein activation. The current proposal will address two issues: (1) how are the AT1-selective ligands guided to the binding pocket in the receptor? and (2) what are the in vivo consequences of the differences between the G-protein mediated and the G-protein independent AT1 receptor signaling? We will determine whether the extracellular loops (ECL) directly participate in the ligand receptor interaction and the activation/inhibition of the AT1 receptor. We anticipate that ECL-2 interacts with the TM domain in an inactive state (R) of the receptor as well as with the Ang II in the activated state (Specific Aim 1). We have discovered that the AT1 receptor engages the beta-arrestin and its fragments in the Ang II activated cells which are absent under other conditions. We will test the hypothesis that site-specific beta-arrestin proteolysis is a novel signaling mode with functional consequences. We will determine the site of beta-arrestin cleavage and its functional consequences in Ang II activated cells (Specific Aim 2). G-protein-dependent AT1 receptor signaling differs from G-protein- independent signaling due to the fact that it causes nuclear translocation of ERK. We will determine the role of Ang ll/AT1 receptor-induced nuclear translocation of ERK 1/2 in activation of gene expression (Specific Aim 3). These studies are essential for a better understanding of the mechanism of Ang II/AT1 receptor action and the development of improved remedies targeting the AT1 receptor.