Store-Operated Ca entry and iPLA2 in vascular SMC

描述（由申请人提供）：我们的长期目标是确定血管平滑肌细胞（SMC）和其他细胞类型中钙池操纵的钙内流（SOCE）的分子机制。最近，我们确定了钙离子非依赖性磷脂酶A2（iPLA 2）作为一种新的决定因素的SOCE，这是成功地证实了他人。我们发现了一种新的质膜界定的级联反应，涉及CIF诱导的抑制性CaM从iPLA 2？的置换，导致其激活和溶血磷脂的产生，进而激活SOC通道。iPLA 2的重要性在最近的果蝇基因筛选中得到了充分的证实，其中iPLA 2 <$，STIM 1和Orai 1（但不是TRP）被发现在SOCE中发挥同样重要的作用，但它们的分子和功能相互作用的机制尚未确定。我们的初步研究强烈表明，iPLA 2 <$可能是STIM 1和Orai 1之间的关键中间体。基于对该领域最新发现的仔细分析，对不同模型的批判性评估以及我们广泛的初步研究结果，我们提出了一种新的SOCE复合物模型（SOCEplex），该模型为SOCE机制带来了新的维度，统一了构象耦合和可扩散信使思想，涉及STIM 1，CIF，iPLA 2？和Orai 1，并提供了新的结构和功能安排。该提议的主要假设是iPLA 2是SOCEplex的中心多功能元件，并作为内质网STIM 1的质膜锚，接受来自耗尽的存储的信号并将信号转换为Orai 1。我们将采用先进的分子、生物化学、影像学、电生理学和功能学方法，在原代血管平滑肌细胞和模型细胞系中测试新的SOCE模型。所有途径和方法都已在PI实验室建立并成功使用。我们的新假设的可行性和拟议研究的成功得到了PI和合作者的广泛初步数据和专业知识的充分支持。该提案的具体目标是：目标1：将iPLA 2 <$确立为STIM 1和Orai 1的分子伴侣，以及SOCEplex的基石。我们将评估SOCE的替代模型，建立iPLA 22作为STIM 1和Orai 1之间的接头的新作用，确定iPLA 2 <$$>与STIM 1相互作用的分子机制及其在SOCE中的作用，确定iPLA 2 <$$>寡聚化的机制，并确定iPLA 2 <$<$中对SOCEplex信号转导至关重要的分子结构域。目的2：探讨CIF和LysoPL在SOCEplex信号转导中的作用。我们将确认CIF在从耗尽的存储到质膜的信号转导中的作用，确定脂筏的作用，并评估LysoPL介导的Orai 1激活机制。公共卫生相关性：我们的长期目标是确定钙库操纵的钙离子进入（SOCE）途径的机制，这是至关重要的各种生理功能。该提案的目标是建立特定的质膜结合的钙离子非依赖性磷脂酶A2亚型作为SOCE机制的重要组成部分的作用，并证明它作为一个中心的分子，结构和功能元件的SOCE复合物（SOCEplex），从耗尽的商店质膜通道的信号转导。我们的新假设的可行性得到了广泛的初步数据和PI实验室先进专业知识的充分支持。

DESCRIPTION (provided by applicant): Our long term goal is to define the molecular mechanism of store-operated Ca2+ entry (SOCE) in vascular smooth muscle cells (SMC) and other cell types. Recently we identified Ca2+-independent phospholipase A2¿ (iPLA2¿) as a novel determinant of SOCE, which was successfully confirmed by others. We discovered a novel plasma membrane-delimited cascade of reactions involving CIF-induced displacement of inhibitory CaM from iPLA2¿ which leads to its activation and production of lysophospholipids that in turn activate SOC channels. The importance of iPLA2¿ was fully confirmed in a recent screen of Drosophila genes, in which iPLA2¿, STIM1 and Orai1 (but not TRP) were found to play equally important roles in SOCE, but the mechanisms of their molecular and functional interactions are yet to be determined. Our preliminary studies strongly suggest that iPLA2¿ may be a key intermediate between STIM1 and Orai1. Based on the careful analysis of the most recent discoveries in the field, critical evaluation of different models, and our extensive preliminary findings, we propose a novel model of SOCE complex (SOCEplex) that brings a new dimension to SOCE mechanism, unifies conformational coupling and diffusible messenger ideas, involves STIM1, CIF, iPLA2¿ and Orai1, and offers their new structural and functional arrangement. The main hypothesis of this proposal is that iPLA2¿ is a central multifunctional element of the SOCEplex, and serves as a plasma membrane anchor for endoplasmic reticulum STIM1, acceptor of the signal from depleted stores and transducer of the signal to Orai1. Novel model of SOCE will be tested in primary vascular SMC and model cell lines using our integrative approach, which involves advanced molecular, biochemical, imaging, electrophysiological and functional methods. All approaches and methods have been established and successfully used in the PI's lab. The feasibility of our new hypothesis and success of the proposed studies are fully supported by extensive preliminary data and expertise of the PI and collaborators. Specific Aims of this proposal are: Aim 1: To establish iPLA2¿ as a molecular partner of STIM1 and Orai1, and a keystone of SOCEplex. We will assess alternative models of SOCE, establish a new role of iPLA22 as a linker between STIM1 and Orai1, identify the molecular mechanism of iPLA2¿ interaction with STIM1 and its role in SOCE, determine the mechanism of iPLA2¿ oligomerization and identify molecular domains in iPLA2¿ that are crucial for signal transduction in SOCEplex. Aim 2: To establish the roles of CIF and LysoPL in signal transduction within SOCEplex. We will confirm the role of CIF in signal transduction from depleted stores to plasma membrane, determine the role of lipid rafts, and assess LysoPL-mediated mechanism of Orai1 activation. PUBLIC HEALTH RELEVANCE: Our long term goal is to define the mechanism of store-operated Ca2+ entry (SOCE) pathway that is crucial for a wide variety of physiological functions. The goals of this proposal are to establish the role of specific plasma membrane-bound isoform of Ca2+-independent phospholipase A2 as a crucial component of SOCE machinery, and to demonstrate that it serves as a central molecular, structural and functional element of SOCE complex (SOCEplex) that transduces the signal from depleted stores to plasma membrane channels. The feasibility of our novel hypothesis is fully supported by extensive preliminary data and advanced expertise of PI's lab.