Store-Operated Ca entry and iPLA2 in vascular SMC

血管 SMC 中存储操作的 Ca 进入和 iPLA2

• 批准号: 7741705
• 负责人: Victoria M Bolotina
• 金额: $ 42.25万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-13 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7741705
• 关键词: Agonist; Agreement; Binding; Biochemical; Blood Vessels; Calcium; Cations; Cell Line; Cell membrane; Cell model; Cells; Complex; Coupling; Data; Dimensions; Drosophila genus; Elements; Endoplasmic Reticulum; Evaluation; Funding; Genes; Goals; Image; Ion Channel; Lysophospholipids; Mediating; Membrane Microdomains; Methods; Modeling; Modification; Molecular; Nitric Oxide; Pathway interactions; Phospholipase; Phospholipase A2; Physiological; Play; Production; Protein Isoforms; Proteins; RNA Editing; Reaction; Relaxation; Research; Role; STIM1 gene; Signal Transduction; Smooth Muscle Myocytes; Testing; Transducers; base; cell type; constriction; molecular domain; novel; programs; public health relevance; success

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.

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Ca2+进入（SOCE）的分子机制。最近我们发现Ca2+独立的磷脂酶A2¿（iPLA2¿）作为SOCE的一个新的决定因素，并被其他人成功证实。我们发现了一种新颖的质膜限定级联反应，涉及 CIF 诱导的 iPLA2 中抑制性 CaM 的置换，从而导致其激活并产生溶血磷脂，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充分证实，其中发现iPLA2¿、STIM1和Orai1（但不是TRP）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 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仔细分析、对不同模型的批判性评估以及我们广泛的初步发现，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的思想，涉及 STIM1、CIF、iPLA2¿ 和 Orai1，并提供了它们新的结构和功能排列。该提议的主要假设是 iPLA2¿ 是 SOCEplex 的中心多功能元件，并充当内质网 STIM1 的质膜锚定物、来自耗尽存储的信号的受体以及将信号转导到 Orai1 的信号。 SOCE 的新模型将使用我们的综合方法在原代血管 SMC 和模型细胞系中进行测试，该方法涉及先进的分子、生化、成像、电生理学和功能方法。所有途径和方法均已在 PI 实验室中建立并成功使用。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 iPLA2 建立为 STIM1 和 Orai1 的分子伴侣，以及 SOCEplex 的基石。我们将评估 SOCE 的替代模型，建立 iPLA22 作为 STIM1 和 Orai1 之间的连接器的新角色，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 寡聚化机制，并确定 iPLA2 中对 SOCEplex 信号转导至关重要的分子结构域。目标 2：确定 CIF 和 LysoPL 在 SOCEplex 信号转导中的作用。我们将确认 CIF 在从耗尽的储存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 激活机制。公共健康相关性：我们的长期目标是确定钙池操纵的 Ca2+ 进入 (SOCE) 途径的机制，该途径对于多种生理功能至关重要。该提案的目标是确定 Ca2+ 独立磷脂酶 A2 的特定质膜结合亚型作为 SOCE 机制的重要组成部分的作用，并证明它作为 SOCE 复合物 (SOCEplex) 的中心分子、结构和功能元件，将信号从耗尽的储存转导到质膜通道。我们的新假设的可行性得到了 PI 实验室广泛的初步数据和先进专业知识的充分支持。