Role of Phospholipase D1 in 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在有利于胞吐的特定位置将囊泡重新聚集到质膜上，然后分阶段融合到质膜上进行的。调节胞吐的全身性缺陷会导致人类的单系统或多系统疾病，调节胞吐的促进(例如在糖尿病中)或抑制(例如在过敏反应中)是许多治疗方法的基础。我们的一般假设是，对iipid环境的操纵是这一过程中的关键因素。已发表的报道和我们的初步证据表明，信号转导酶磷脂酶D1(PLD1)在这一过程中发挥了后期作用，通过产生磷脂酸(PA)将小泡融合到质膜中。许多调查途径现在适时地进行探索，以确定它的运作机制。我们建议开展以下具体目标来解决这些问题：1.PLD1的激活和PA的产生与它们促进调控胞吐的时间和空间关系是什么？我们将检查在分泌时是否迫切需要PLD1激活，哪些PLDV激活剂参与了PLD1促进的过程，以及在融合事件中PA在哪里产生。2.增加PA水平是如何促进调节胞吐作用的？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的形态，以确定融合过程被阻止在哪一步。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)调节PI4，5P2的产生；募集到CAPS的胞外融合位点，这是融合所需的一种PI4，5P2结合蛋白；以及通过促进融合孔的形成或扩张对融合过程本身的潜在影响。拟议的实验结果将大大加深我们对PLD1在调节胞吐过程中促进分泌的机制的理解。

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.