Role of Phospholipase D1 in regulated exocytosis

磷脂酶 D1 在调节胞吐作用中的作用

• 批准号: 7036416
• 负责人: Michael A. Frohman
• 金额: $ 29.12万
• 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: 2006-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7036416
• 关键词: RNA interference; adipocytes; alcohol phosphotransferase; biological signal transduction; calcium binding protein; cell line; chromaffin cells; electron microscopy; enzyme activity; enzyme mechanism; exocytosis; guanine nucleotide binding protein; membrane fusion; pancreatic islets; phosphatidate; phosphatidylinositols; phospholipase D; protein kinase C; protein protein interaction; transfection /expression vector; vesicle /vacuole

DESCRIPTION (provided by applicant): Regulated exocytosis of secretory membrane vesicles proceeds via recruitment of the vesicles to the plasma membrane at specific sites conducive for exocytosis, followed by staged fusion into the plasma membrane. Generalized defects in regulated exocytosis cause single or multi-system disease in humans, and the promotion (e.g in diabetes) or inhibition (e.g. in anaphylaxis) of regulated exocytosis underlies numerous therapeutic modalities. Our general hypothesis is that manipulation of the iipid environment is a key element in this process. Published reports and our preliminary evidence suggest that the signal-transducing enzyme Phospholipase D1 (PLD1) plays a role late in this process during fusion of the vesicles into the plasma membrane via production of phosphatidic acid (PA), the Iipid product of PLD action. Many avenues of investigation are now timely to explore to determine the mechanism through which it functions. We propose to carry out the following specific aims to address these questions: 1. What are the temporal and spatial relationships of PLD1 activation and PA generation to their facilitation of regulated exocytosis? We will examine whether PLD1 activation is required acutely at the time of secretion, which of PLDVs activators participate in the PLD1-facilitated process, and where PA is produced during the fusion event. 2. How is regulated exocytosis facilitated by increasing levels of PA? We will use electron microscopy to examine the morphology of vesicles hindered from completing fusion in cells lacking PLD1 to determine which step the fusion process is blocked at. We will also examine potential mechanisms though which PLD1 and its product PA may be functioning, including regulation of the production of PI4,5P2 by stimulation of PI4P5KI, the enzyme family that generates PI4,5P2; recruitment to exocytic fusion sites of CAPS, a PI4,5P2-binding protein that is required for fusion; and potential affects on the fusion process itself though promotion of fusion pore formation or expansion. The results from the proposed experiments will substantially further our understanding of the mechanisms through which PLD1 promotes secretion during regulated exocytosis.

描述（由申请人提供）：分泌膜囊泡的调节胞吐作用是通过将囊泡募集到有利于胞吐作用的特定部位的质膜，然后将融合到质膜中。受调节性胞吐作用的普遍缺陷导致人类中的单一或多系统疾病，以及受调节胞吞作用的促进（例如，在糖尿病中）或抑制（例如在过敏反应中）是许多治疗方式的基础。我们的总体假设是，对IIPID环境的操纵是此过程中的关键要素。发表的报告和我们的初步证据表明，在囊泡融合到质膜中，通过生产磷脂酸（PA），PLD作用的IIPID产物在囊泡中融合到质膜中，在此过程中，信号转移磷脂酶D1（PLD1）在此过程中起作用。现在，许多调查途径及时探索以确定其功能的机制。我们建议执行以下具体目的解决以下问题：1。PLD1激活和PA生成与促进受调节胞吐作用的时间和空间关系是什么？我们将检查在分泌时是否需要急性激活PLD1激活，PLDVS激活剂的哪个参与PLD1-核酸过程，以及在融合事件中产生PA的位置。 2。如何通过升高的PA水平促进受调节的胞吐作用？我们将使用电子显微镜检查缺少PLD1的细胞中融合的囊泡的形态，以确定融合过程被阻断的哪个步骤。我们还将检查潜在的机制，尽管PLD1及其产物PA可能起作用，包括通过刺激PI4P5KI（生成PI4,5P2）的PI4P5KI来调节PI4,5P2的产生；募集到Caps的胞外融合位点，Caps是一种融合所需的PI4,5P2结合蛋白；通过促进融合孔形成或扩展，潜在影响融合过程本身。提出的实验的结果将大大进一步了解PLD1在调节胞吐作用过程中促进分泌的机制。