Roles of Phospholipase D in AT1 Receptor Endocytosis

磷脂酶 D 在 AT1 受体胞吞作用中的作用

• 批准号: 7045915
• 负责人: GUANGWEI DU
• 金额: $ 27.04万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7045915
• 关键词: actins; angiotensin II; angiotensin receptor; biological signal transduction; cell line; clathrin; enzyme activity; enzyme induction /repression; intermolecular interaction; intracellular transport; lipid raft; membrane structure; molecular site; phosphatidate; phosphatidylinositols; phospholipase D; protein localization; protein transport; receptor mediated endocytosis; small interfering RNA; transmission electron microscopy

DESCRIPTION (provided by applicant): The angiotensin II type I receptor (AT1R) mediates diverse intracellular responses to angiotensin II (Ang II) in the regulation of blood pressure, hydromineral balance, cardiac hypertrophy and cell proliferation. Endocytosis of the Ang ll-bound receptor not only desensitizes AT1R signaling from the cell surface but also activates specific intracellular signaling pathways. The long-term goal of this project is to elucidate the signaling pathways that regulate AT1R subcellular trafficking. Clarifying the events that ensue will potentially suggest approaches for novel therapeutics for cardiovascular diseases related to AT1R dysfunction, and will provide new insights into the mechanism underlying receptor endocytosis in general. Although roles for many proteins in AT1R trafficking are becoming clear, less is known about the specific functions of membrane lipids. It has been recognized that interaction between proteins and membrane (particularly phospholipids) drives initiation, fission, and fusion of endocytic vesicles. However, how phospholipid metabolism is regulated during this process and, in turn, triggers signals for the different receptor endocytic steps, remains unclear. The Phospholipase D (PLD) family contains two members, PLD1 and PLD2, both of which catalyze the hydrolysis of phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline. PLD2-generated PA has been proposed to regulate a number of endocytic and actin regulatory proteins and several types of membrane trafficking processes. The hypothesis of this proposal, based on our published work and preliminary evidence, is that PLD2-generated PA plays a central signaling role in regulation of AT1R endocytosis and signaling. In specific aim 1, we will test the hypothesis that PLD2 facilitates AT1R endocytosis by regulating spatiotemporal dynamics of phosphatidylinositol 4,5-bisphosphate {PI(4,5)P2}, which is an essential phospholipid in endocytosis. Specific aim 2 will test whether PLD2 regulates formation of clathrin-coated pits (CCPs) by determining which step in CCP formation is blocked when PLD2 is downregulated by small hairpin RNA (shRNA). We will also examine whether membrane microdomains regulated by PLD2 signaling are critical for CCP formation. Specific aim 3 will test whether differential distribution of AT1R as regulated by PLD2 determines the output of AT1R signaling. AT1R activation in different subcellular locations results in distinct signaling outputs. We will determine whether PLD2 downregulation changes the kinetic and spatial patterns of Ang II signaling, and as a result, affects cell growth.

描述（由申请人提供）：血管紧张素II I型受体（AT1R）介导多种细胞内对血管紧张素II（Ang II）的反应，调节血压、水矿物质平衡、心脏肥大和细胞增殖。 Ang II结合受体的内吞作用不仅使来自细胞表面的AT1R信号传导脱敏，而且还激活特定的细胞内信号传导途径。该项目的长期目标是阐明调节 AT1R 亚细胞运输的信号通路。澄清随后发生的事件可能会为与 AT1R 功能障碍相关的心血管疾病提供新的治疗方法，并将为受体内吞作用的一般机制提供新的见解。尽管许多蛋白质在 AT1R 运输中的作用越来越清楚，但人们对膜脂的具体功能知之甚少。人们已经认识到蛋白质和膜（特别是磷脂）之间的相互作用驱动内吞囊泡的起始、裂变和融合。然而，在此过程中如何调节磷脂代谢并进而触发不同受体内吞步骤的信号仍不清楚。磷脂酶 D (PLD) 家族包含两个成员，PLD1 和 PLD2，两者均催化磷脂酰胆碱 (PC) 水解生成磷脂酸 (PA) 和胆碱。 PLD2 产生的 PA 被认为可以调节许多内吞和肌动蛋白调节蛋白以及几种类型的膜运输过程。基于我们发表的工作和初步证据，该提案的假设是 PLD2 生成的 PA 在 AT1R 内吞作用和信号传导的调节中发挥着核心信号传导作用。在具体目标 1 中，我们将检验以下假设：PLD2 通过调节磷脂酰肌醇 4,5-二磷酸 {PI(4,5)P2}（内吞作用中必需的磷脂）的时空动力学来促进 AT1R 内吞作用。具体目标 2 将通过确定当 PLD2 被小发夹 RNA (shRNA) 下调时 CCP 形成中的哪一步被阻断来测试 PLD2 是否调节网格蛋白包被的凹坑 (CCP) 的形成。我们还将研究 PLD2 信号传导调节的膜微域是否对 CCP 形成至关重要。具体目标3将测试PLD2调节的AT1R的差异分布是否决定AT1R信号的输出。不同亚细胞位置的 AT1R 激活会产生不同的信号输出。我们将确定 PLD2 下调是否会改变 Ang II 信号传导的动力学和空间模式，从而影响细胞生长。