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)和其他细胞类型中存储操作的钙内流(SOCE)的分子机制。最近，我们发现钙非依赖性磷脂酶A2(IPLA2)是SOCE的一个新的决定因素，并被其他人成功地证实。我们发现了一种新的质膜限定的级联反应，涉及CIF诱导抑制CaM从iPLA2[1]置换，导致其激活和产生溶血磷脂，进而激活SOC通道。IPLA2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iPLA2、STIM1和Orai1(而不是Trp)在SOCE中起着同样重要的作用，但它们的分子和功能相互作用的机制尚未确定。我们的初步研究有力地表明，iPLA2可能是STIM1和Orai1之间的关键中间体。基于对该领域最新发现的认真分析、对不同模型的评价以及我们广泛的初步发现，我们提出了一个新的SOCE复合体模型(SOCEplex)，该模型为SOCE机制带来了一个新的维度，统一了构象耦合和扩散信使的思想，涉及STIM1、CIF、iPLA2和Orai1，并给出了它们新的结构和功能安排。这一设想的主要假设是，iPLA2是SOCEplex的一个中枢多功能元件，是内质网STIM1的质膜锚，是耗竭储备物信号的受体，也是Orai1信号的转导者。新的SOCE模型将在原代血管SMC和模型细胞系中使用我们的综合方法进行测试，该方法涉及先进的分子、生化、成像、电生理和功能方法。所有的途径和方法都已经建立，并成功地在PI的实验室中使用。我们新假设的可行性和拟议研究的成功完全得到了PI和合作者的大量初步数据和专业知识的支持。这项提议的具体目标是：目标1：建立iPLA2？作为STIM1和Orai1的分子伙伴和SOCEplex的基石。我们将评估不同的SOCE模型，建立iPLA22作为STIM1和Orai1之间连接物的新角色，确定iPLA2与STIM1相互作用的分子机制及其在SOCE中的作用，确定iPLA2齐聚的机制，并确定iPLA2中对SOCEplex中的信号转导至关重要的分子结构域。目的：探讨CIF和LysoPL在SOCEplex信号转导中的作用。我们将确认CIF在从耗尽的贮备物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Orai1激活的机制。与公共健康相关：我们的长期目标是确定对多种生理功能至关重要的存储操作的钙离子进入(SOCE)途径的机制。这项建议的目的是确定特定的质膜结合的钙非依赖性磷脂酶A2作为SOCE机制的重要组成部分的作用，并证明它是SOCE复合体(SOCEplex)的中心分子、结构和功能元件，将信号从耗尽的存储传递到质膜通道。我们的新假设的可行性得到了广泛的初步数据和Pi实验室的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.