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通过将囊泡募集到质膜上有利于胞吐作用的特定位点进行，然后分阶段融合到质膜中。调节性胞吐作用的普遍缺陷会导致人类单系统或多系统疾病，而调节性胞吐作用的促进（例如糖尿病）或抑制（例如过敏反应）是许多治疗方式的基础。我们的一般假设是，对 iipid 环境的操纵是这个过程中的关键因素。已发表的报告和我们的初步证据表明，信号转导酶磷脂酶 D1 (PLD1) 在囊泡通过产生磷脂酸 (PA)（PLD 作用的脂质产物）融合到质膜的过程中发挥了重要作用。现在需要及时探索许多调查途径，以确定其发挥作用的机制。我们建议实现以下具体目标来解决这些问题： 1. PLD1 激活和 PA 生成与其促进受调节的胞吐作用的时间和空间关系是什么？我们将检查分泌时是否需要急速激活 PLD1、哪些 PLDV 激活剂参与 PLD1 促进过程以及融合过程中 PA 是在哪里产生的。 2. 增加 PA 水平如何促进受调节的胞吐作用？我们将使用电子显微镜检查缺乏 PLD1 的细胞中阻碍完成融合的囊泡形态，以确定融合过程在哪一步被阻断。我们还将研究 PLD1 及其产物 PA 可能发挥作用的潜在机制，包括通过刺激 PI4P5KI（产生 PI4,5P2 的酶家族）来调节 PI4,5P2 的产生；募集到 CAPS 的胞吐融合位点，CAPS 是融合所需的 PI4,5P2 结合蛋白；通过促进熔合孔的形成或扩张对熔合过程本身产生潜在影响。所提出的实验结果将大大加深我们对 PLD1 在受调节的胞吐作用期间促进分泌的机制的理解。