Role of Gbeta 2 in the Nucleus of Angiotensin Receptor Activated Cells

描述(申请人提供)：肾素-血管紧张素系统激素，血管紧张素II(Ang II)是正常心血管生理的主要调节因子。正常情况下，Ang II调节血压和水电解质平衡的急性变化。在病理条件下，Ang II调节基因表达，导致靶细胞重塑程序。两种类型的血管紧张素Ⅱ的作用都是由AT1受体介导的。AT1受体拮抗剂用于抗高血压治疗，目前正在进行临床试验，以预防充血性心力衰竭和其他类型的终末器官损害。这些药物除了控制Ang II的急性作用外，还逆转重塑基因表达的变化。因此，从机制上了解AT1受体对基因表达的调控是至关重要的。我们通过跟踪血管紧张素Ⅱ受体激活的细胞核室中蛋白质之间的相互作用来解决这个问题。为了确定参与基因表达调控的新的蛋白质复合体，我们用高通量质谱仪分析了AT1受体激活的细胞的核蛋白质组。在转移到细胞核的候选蛋白质中，G蛋白Beta2(G(32)亚基多肽是一个令人惊讶的发现。GP是异三聚体G蛋白的一种成分，G蛋白是GPCR信号的重要转导分子。GPCR的激活，如血管紧张素Ⅱ结合时的AT1受体，导致G蛋白催化解离成GPY和GOT亚基，进而激活它们各自的效应器。Gp和Gy亚单位总是保持在复合体中，它们通常通过对Gy射线的脂质修饰而拴在质膜内表面。因此，传统上，GP被认为是在质膜上传递信号，从未被证明是核蛋白质组的一部分。我们发现，当AT1受体激活时，GP2移位到细胞核中，并与HDAC5和α-肌动蛋白-4形成复合体。肌动蛋白-4是一种钙结合蛋白，HDAC5是一种染色质重塑酶。因此，我们假设血管紧张素II激活的AT1受体刺激GP的核移位和涉及肌动蛋白-4和HDAC5的钙敏感染色质重塑复合体的组装。本项目的目标是(I)通过突变和Biaccore分析来确定这三个分子之间相互作用的特异性，以及(Ii)通过微阵列分析细胞中Gbeta2的RNAi下调中AT1受体依赖的基因表达来确定这种复合体形成的功能意义。我们提出的研究将展示一种通过GPCR控制基因表达的新模式。

DESCRIPTION (provided by applicant): The rennin-angiotensin system hormone, angiotensin II (Ang II) is a master regulator of normal cardiovascular physiology. Normally, Ang II regulates acute changes in blood pressure and water-electrolyte balance. Under pathological conditions, Ang II modulates gene expression leading to a remodeling program in the target cells. Both types of Ang II actions are mediated by AT1 receptors. Antagonists of AT1 receptor are used in antihypertensive therapy and are currently in clinical trial for prevention of congestive heart failure and other types of end organ damage. These drugs reverse the remodeling gene expression changes in addition to controlling the acute actions of Ang II. Therefore, a mechanistic understanding of modulation of gene expression by AT1 receptor is essential. We approached this problem by tracking protein-protein interactions in the nuclear compartment of Ang II receptor activated cells. To identify novel protein complexes involved in the modulation of gene expression, we analyzed nuclear proteome of AT1 receptor-activated cells by high throughput mass-spectrometry. Among the candidate proteins that translocated to the nucleus, the G-protein beta2 (G(32) subunit polypeptide was a surprising finding. GP is a component of heterotrimeric G proteins which are vital transducers of GPCR signals. Activation of a GPCR, such as AT1 receptor upon Ang II binding, causes catalytic dissociation of G-proteins into Gpy and Got subunits which in turn activate their respective effectors. The GP and Gy subunits always remain in complex and they are generally tethered to the inner face of the plasma membrane by lipid modifications on Gy. Therefore, the GP has traditionally been thought to transduce signals at plasma membrane and has never been shown to be part of the nuclear proteome. We found that Gp2 translocated into the nucleus upon AT1 receptor activation and formed a complex with HDAC5 and alpha-actinin-4. Actinin-4 is a calcium binding protein and HDAC5 is a chromatin remodeling enzyme. Hence, we hypothesize that Ang II activated AT1 receptor stimulates nuclear translocation of GP and assembly of a calcium sensitive chromatin remodeling complex involving actinin-4 and HDAC5. The goals of this project are (i) to determine the specificity of interaction between these three molecules using mutagenesis and Biacore analysis and (ii) to establish the functional significance of this complex formation by microarray analysis of AT1 receptor-dependent gene expression in RNAi knock-down of Gbeta 2 in cells. Our proposed studies will demonstrate a novel mode of control of gene expression by a GPCR.