Roles of Phospholipase D in AT1 Receptor Endocytosis

血管紧张素II I型受体（AT1R）介导对血管紧张素II的多种细胞内反应 (Ang II）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细胞表面的AT1R信号转导脱敏， 激活特定的细胞内信号通路。本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 提出了用于与AT1R功能障碍相关的心血管疾病的新疗法的方法，并且将 为受体内吞作用的机制提供了新的见解。 虽然许多蛋白质在AT1R运输中的作用越来越清楚，但对AT1R的作用知之甚少。 膜脂的特殊功能。已经认识到，蛋白质和 膜（特别是磷脂）驱动内吞囊泡的起始、分裂和融合。然而，在这方面， 磷脂代谢在此过程中是如何调节的，反过来，触发了不同的信号。 受体内吞步骤，仍然不清楚。磷脂酶D（PLD）家族包含两个成员，PLD 1 和PLD 2，它们都催化磷脂酰胆碱（PC）水解生成磷脂酸 (PA)和胆碱。已经提出PLD 2产生的PA调节许多内吞和肌动蛋白， 调节蛋白和几种类型的膜运输过程。这个提议的假设， 基于我们已发表的工作和初步证据，PLD2产生的PA发挥着中枢信号作用， 在调节AT1R内吞和信号传导中的作用。 在具体的目标1中，我们将检验PLD 2通过调节AT1R内吞促进AT1R内吞的假设。 磷脂酰肌醇4，5-二磷酸{PI（4，5）P2}的时空动力学，这是一个重要的 内吞作用中的磷脂。具体目标2将测试PLD 2是否调节网格蛋白包被的凹坑的形成 通过确定当PLD2被小分子抑制剂下调时，CCP形成中的哪一步被阻断， 发夹RNA（shRNA）。我们还将研究膜微结构域是否受PLD2信号转导的调节， 对CCP的形成至关重要。具体目标3将测试AT1R的差异分布是否受 PLD 2确定AT1R信令的输出。不同亚细胞位置的AT1R激活导致 不同的信号输出。我们将确定PLD 2下调是否改变了细胞的动力学和空间分布。 血管紧张素II信号的模式，并因此影响细胞生长。

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 wilt 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) drive 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.