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

描述（由申请人提供）：肾素-血管紧张素系统激素，血管紧张素II（Ang II）是正常心血管生理学的主要调节剂。正常情况下，血管紧张素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，导致靶细胞中的重塑程序。这两种类型的血管紧张素II的行动是由AT 1受体介导的。AT 1受体拮抗剂用于抗高血压治疗，目前正在进行预防充血性心力衰竭和其他类型终末器官损伤的临床试验。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，了解AT 1受体对基因表达的调节机制是至关重要的。我们通过跟踪血管紧张素II受体激活细胞核区室中的蛋白质-蛋白质相互作用来解决这个问题。为了鉴定参与基因表达调控的新型蛋白质复合物，我们通过高通量质谱分析了AT 1受体激活细胞的核蛋白质组。在转移到细胞核的候选蛋白质中，G蛋白β 2（G（32））亚基多肽是一个令人惊讶的发现。GP是异源三聚体G蛋白的组成部分，异源三聚体G蛋白是GPCR信号的重要转导子。在Ang II结合时，GPCR（例如AT 1受体）的激活导致G蛋白催化解离成Gpy和Got亚基，Gpy和Got亚基又激活它们各自的效应物。GP和戈伊亚基总是保持复合体，并且它们通常通过戈伊上的脂质修饰而被束缚在质膜的内表面。因此，GP传统上被认为在质膜上传递信号，并且从未被证明是核蛋白质组的一部分。我们发现Gp 2在AT 1受体激活后易位到细胞核中，并与HDAC 5和α-辅肌动蛋白-4形成复合物。肌动蛋白-4是钙结合蛋白，HDAC 5是染色质重塑酶。因此，我们推测，血管紧张素II激活AT 1受体刺激核转位的GP和装配的钙敏感性染色质重塑复合物涉及辅肌动蛋白-4和HDAC 5。该项目的目标是（i）使用诱变和Biacore分析来确定这三种分子之间相互作用的特异性，以及（ii）通过在细胞中RNAi敲低G β 2中AT 1受体依赖性基因表达的微阵列分析来确定这种复合物形成的功能意义。我们提出的研究将证明一种新的模式控制基因表达的GPCR。

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.