Roles of Phospholipase D in AT1 Receptor Endocytosis

描述（由申请人提供）：血管紧张素II I型受体（AT1R）介导血管紧张素II （Ang II）的多种细胞内反应，调节血压、水矿物质平衡、心脏肥厚和细胞增殖。Ang - ll结合受体的内吞作用不仅使来自细胞表面的AT1R信号脱敏，而且激活特定的细胞内信号通路。该项目的长期目标是阐明调节AT1R亚细胞运输的信号通路。阐明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，并将为一般受体内吞作用的机制提供新的见解。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了内吞囊泡的起始、裂变和融合。然而，在这一过程中磷脂代谢是如何被调节的，以及如何触发不同受体内吞步骤的信号，目前尚不清楚。磷脂酶D （Phospholipase D， PLD）家族包含PLD1和PLD2两个成员，它们都催化磷脂酰胆碱（phosphatidyolcholine， PC）水解生成磷脂酸（phosphatidic acid， PA）和胆碱。pld2产生的PA已被提出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。基于我们已发表的工作和初步证据，本提案的假设是pld2产生的PA在AT1R内吞和信号传导的调节中起着核心的信号传导作用。在具体目标1中，我们将验证PLD2通过调节磷脂酰肌醇4,5-二磷酸{PI(4,5)P2}的时空动态来促进AT1R内吞的假设，这是内吞作用中必需的磷脂。特异性目标2将通过确定当PLD2被小发夹RNA （shRNA）下调时CCP形成的哪个步骤被阻断，来测试PLD2是否调节网格蛋白包被坑（CCP）的形成。我们还将研究由PLD2信号调节的膜微域是否对CCP的形成至关重要。具体目标3将测试PLD2调节的AT1R的差分分布是否决定了AT1R信号的输出。AT1R在不同亚细胞位置的激活导致不同的信号输出。我们将确定PLD2下调是否会改变Ang II信号的动力学和空间模式，从而影响细胞生长。

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.