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）和其他细胞类型中储存操作的Ca2+进入（SOCE）的分子机制。最近，我们发现Ca2+独立磷脂酶A2¿（iPLA2¿）是SOCE的一个新的决定因素，这已被其他人成功证实。我们发现了一种新的质膜划定的级联反应，涉及cif诱导iPLA2¿中抑制性CaM的位移，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证实，其中iPLA2¿、STIM1和Orai1（但不包括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，我们提出了一个新的SOCE复合物模型（SOCEplex），该模型为SOCE机制带来了新的维度，统一了构象耦合和扩散信使思想，涉及STIM1， CIF， iPLA2¿和Orai1，并提供了新的结构和功能安排。本研究的主要假设是iPLA2¿是SOCEplex的核心多功能元件，并作为内质网STIM1的质膜锚点，STIM1是来自耗尽存储的信号的受体和信号到Orai1的换能器。我们将采用先进的分子、生化、成像、电生理和功能等综合方法，在原发性血管SMC和模型细胞系中测试新型SOCE模型。所有的方法和方法都已经建立并成功地在PI的实验室中使用。我们的新假设的可行性和提议的研究的成功完全得到了广泛的初步数据和PI和合作者的专业知识的支持。本提案的具体目标是：目标1：建立iPLA2¿作为STIM1和Orai1的分子合作伙伴和SOCEplex的基石。我们将评估SOCE的替代模型，建立iPLA22作为STIM1和Orai1之间的连接物的新作用，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寡聚化的机制，并确定iPLA2¿中对SOCEplex信号转导至关重要的分子结构域。目的2：建立CIF和LysoPL在SOCEplex信号转导中的作用。我们将确认CIF在从耗尽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。公共卫生相关性：我们的长期目标是确定存储操作的Ca2+进入（SOCE）途径的机制，这对多种生理功能至关重要。本提案的目标是确定Ca2+非依赖性磷脂酶A2的特定质膜结合异构体作为SOCE机制的关键组成部分的作用，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广泛的初步数据和PI实验室先进的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.