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: 7584587
• 负责人: Victoria M Bolotina
• 金额: $ 41.88万
• 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/7584587
• 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（由适用提供）：我们的长期目标是定义血管平滑肌细胞（SMC）和其他细胞类型中商店操作的Ca2+进入（SOCE）的分子机制。最近，我们将Ca2+独立的磷脂酶A2（IPLA2¿）确定为对SOCE的一种新颖，这是他人成功证实的。我们发现了一种新型的质膜脱落的反应级联反应，涉及CIF引起的抑制性凸轮从IPLA2¿中移位，这导致其激活和产生溶血脂，从而激活SOC通道。在最近的果蝇基因的筛选中，IPLA2¿的重要性得到了充分的证实，其中IPLA2¿，STIM1和ORAI1（而不是TRP）在SOCE中起着同样重要的作用，但其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。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，我们提出了一种新颖的Soce Complex（SocePlex）模型，为SOCE机制带来了新的维度，UnifieDs会议耦合和散布的Messenger想法涉及Stim1，CIF，CIF，IPLA2，并提供了新的结构，并提供了新的结构。该提案的主要假设是IPLA2¿是Soceplex的中心多功能元素，它是内质网的质膜锚固剂，用于内质网刺激器，是信号耗尽商店和信号传感器信号的信号的受体。 SOCE的新型模型将使用我们的集成方法在原代血管SMC和模型细胞系中进行测试，该方法涉及晚期分子，生化，成像，电生理和功能方法。所有方法和方法均已在PI实验室中建立并成功使用。我们的新假设和拟议研究成功的可行性得到了PI和合作者的广泛初步数据和专业知识的充分支持。该提案的具体目的是：目标1：建立IPLA2€作为STIM1和ORAI1的分子伴侣，以及Soceplex的基石。 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¿ Aim 2: To establish the roles of CIF and LysoPL in signal transduction within SOCEplex.我们将确认CIF在从枯竭存储到质膜的信号转导中的作用，确定脂质筏的作用，并评估溶酶体介导的ORAI1激活机制。公共卫生相关性：我们的长期目标是定义商店经营的CA2+进入（SOCE）途径的机制，这对于各种物理功能至关重要。该提案的目标是确定Ca2+非依赖性磷脂酶A2特定质膜结合的同工型的作用，作为SOCE机械的关键组成部分，并证明它是So​​ce Compeffer（SoCePelex）的中心分子，结构和功能元素（SOCEPELX），从而将信号转换为Depleted StoreSmbrane Membrane pmbrane pmbrane to plasembrane。我们新颖的假设的可行性得到了广泛的初步数据和PI实验室的高级专业知识的充分支持。