Calcium Signaling Roles of STIM1 and STIM2 in Smooth Muscle

描述（由申请人提供）：钙信号在控制细胞事件中至关重要。在平滑肌细胞（SMC）中，短期Ca 2+信号控制收缩反应，而长期Ca 2+信号通过Ca 2+介导的转录控制来调节细胞生长和增殖。拟议的研究集中在两个中心重要的Ca 2+信号转导，STIM 1和STIM 2。STIM蛋白是精细调节的内质网（ER）Ca 2+传感器。STIM蛋白被触发以自缔合并移位到专门的ER-PM连接中，在该连接中，STIM蛋白门控高度Ca 2+选择性的“储存操纵的”奥赖通道。STIM蛋白还靶向“电压操纵”L型Ca 2+通道（LTCC），并对这两个通道靶点施加相互控制，激活奥赖但失活LTCC，这可能对SMC生长表型变化很重要，其中LTCC丢失，STIM-奥赖信号传导占主导地位。虽然STIM 1已被深入研究，但一个主要的挑战是了解研究不足的STIM 2蛋白的作用：其结构，功能和表达的差异如何导致强大的信号传导和表型变化。尽管STIM 1或STIM 2的整个动物敲除是致命的，但我们在小鼠中产生了STIM 1、STIM 2和STIM+ STIM 2的条件性SMC靶向缺失。SMC特异性STIM 1-KO导致发育不良的动物过早死亡，SMC在体内和体外经历生长转变的能力存在重大缺陷。SMC条件性STIM 1/STIM 2双敲除是围产期致死的，表明STIM 1和STIM 2在SMC功能和生长转变中的重要而独特的作用。使用这些系统，我们有两个独立但相互依赖的具体目标1。了解STIM 1和STIM 2激活的机制和区别，并确定它们与靶向Ca 2+通道的分子偶联。使用突变修饰，高分辨率的Ca 2+和FRET成像，和电生理学，我们的目标是定义传感和目标通道耦合的分子基础STIM 1，定义机械重要的区别，在研究不足的STIM 2蛋白。基于STIM 1和STIM 2之间新的结构见解和主要功能差异，我们的研究重点是定义两种STIM蛋白与奥赖和LTCC靶通道相互作用并对其进行门控的相互作用。2.了解STIM 1和STIM 2在丝裂原诱导的SMC从静止向增殖转化中的不同作用。使用我们的SMC靶向STIM 1和STIM 2空背景小鼠以及分子探针来特异性修饰STIM 1和STIM 2介导的靶通道偶联，我们的目的是确定STIM 1和STIM 2在介导SMC生长转变中的差异有效性。研究将确定STIM 1和STIM 2表达的变化以及它们的激活和通道耦合的区别如何导致Ca 2+信号，NFAT激活和改变生长过渡的不同特征。我们的研究适用于：（a）关于STIM蛋白的结构和通道偶联的新知识，（B）创新的STIM 1和STIM 2基因缺失动物模型，（c）评估STIM 1和STIM 2靶通道偶联的创新探针-提供对STIM 1在控制SMC生长转变中的关键作用的新理解。我们的目标还解决了关于了解STIM机制、行动和作用的信息基本缺乏的问题2。这些研究的信息对于了解血管SMC损伤反应如何发生以及如何控制至关重要。我们的模型预测，不同模式的STIM 1和STIM 2的表达可以是一个决定因素，在SMC是否经历表型变化。因此，这项工作在理解和预防SMC生长变化方面具有根本的重要性，SMC生长变化是主要血管疾病（包括动脉粥样硬化、高血压和动脉再狭窄）的基础，SMC表型变化是哮喘和支持肿瘤生长的血管生成中肺反应的基础。

DESCRIPTION (provided by applicant): Calcium signals are vital in controlling cellular events. In smooth muscle cells (SMC), short term Ca2+ signals control contractile responses whereas longer term Ca2+ signals regulate cell growth and proliferation through Ca2+-mediated transcriptional control. The proposed studies focus on the two centrally important Ca2+ signal transducers, STIM1 and STIM2. STIM proteins are finely-tuned endoplasmic reticulum (ER) Ca2+ sensors. STIM proteins are triggered to self-associate and translocate into specialized ER-PM junctions within whom the STIM proteins gate the highly Ca2+ selective "store-operated" Orai channels. STIM proteins also target "voltage-operated" L-type Ca2+ channels (LTCC) and exert reciprocal control over these two channel targets, activating Orai but deactivating LTCC, likely important to SMC growth phenotype change in which LTCC is lost and STIM-Orai signaling predominates. While STIM1 has been intensively examined, a major challenge is to understand the role of the poorly-studied STIM2 protein: how differences in its structure, function and expression lead to powerful signaling and phenotypic changes. While whole animal knockout of either STIM1 or STIM2 are lethal, we generated conditional SMC-targeted deletions of STIM1, STIM2, and STIM+STIM2 in mice. The SMC-specific STIM1-KO results in poorly-developed animals dying early, with major defects in the ability of SMC to undergo growth transition, in vivo and in vitro. The SMC conditional STIM1/STIM2 double knockout is perinatally lethal indicating important yet distinct roles of STIM1 and STIM2 in SMC function and growth transition. Using these systems, we have two independent but inter-dependent specific aims 1. To understand the mechanisms and distinctions between STIM1 and STIM2 activation, and to define their molecular coupling to target Ca2+ channels. Using mutational modifications, high resolution Ca2+ and FRET imaging, and electrophysiology, we aim to define the molecular basis of sensing and target channel coupling for STIM1, defining mechanistically important distinctions in the poorly studied STIM2 protein. Building on new structural insights and major functional distinctions between STIM1 and STIM2, our studies focus on defining the interactions through which the two STIM proteins interact with and gate Orai and LTCC target channels. 2. To understand the distinct roles of STIM1 and STIM2 in mediating mitogen-induced growth transition of SMC from the quiescent to proliferative phenotype. Using our SMC-targeted STIM1 and STIM2 null-back- ground mice together with molecular probes to specifically modify STIM1- and STIM2-mediated target channel coupling, we aim to define the differential effectiveness of STIM1 and STIM2 in mediating SMC growth transition. Studies will determine how changes in STIM1 and STIM2 expression and distinctions in their activation and channel coupling, lead to distinct profiles of Ca2+ signals, NFAT activation, and altered growth transition. Our studies apply: (a) new knowledge on the structure and channel-coupling of STIM proteins, (b) innovative STIM1 and STIM2 gene-deletion animal models, (c) innovative probes to assess STIM1 and STIM2 target channel coupling - to provide new understanding of the crucial role of STIM1 in controlling SMC growth transition. Our goals also address a fundamental paucity of information on understanding the mechanism, action and role of STIM2. Information from these studies is crucial to understanding how vascular SMC injury responses occur and how they may be controlled. Our model predicts that distinct patterns of STIM1 and STIM2 expression can be a determining factor in whether SMC undergo phenotype change. Hence the work has fundamental importance in understanding and preventing the SMC growth changes that underlie major vascular diseases including atherosclerosis, hypertension, and arterial restenosis, and SMC phenotype change that underlies lung responses in asthma and angiogenesis that supports growth of tumors.