Role of caveolae in G protein signaling

小凹在 G 蛋白信号传导中的作用

• 批准号: 9210145
• 负责人: Suzanne F Scarlata
• 金额: $ 24.29万
• 依托单位: WORCESTER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9210145
• 关键词: Role; caveolae; protein; signaling

 DESCRIPTION (provided by applicant): Muscle cells undergo both healthy and unhealthy adaptive changes in response to environmental cues. A notable example is the hypertrophic growth of cardiac myocytes following infarction. Cardiac hypertrophy is known to be regulated in some way through the Gq family of G proteins which mediates signals from hypertrophic agents such as angiotensin II. Gq activates phospholipase C- (PLC) enzymes to produce Ca2+ signals in cells. Long term adaptive Gq responses involve Rho/Rac protein. We have shown that the activation state of Gq is stabilized by caveolae. Caveolae are membrane invaginations that can mechanically accommodate increases in cell volume by flattening. We find that this flattening releases Gq and destabilizes its association with PLC. Ca2+ signals are then attenuated. Reduced association with Gq allows PLC to move into the cytosol to bind to C3PO. C3PO is required for efficient RNA silencing of gene by promoting the activity of the RNA-induced silencing complex (RISC). We have found that increasing PLC reverses RN A silencing of specific genes through its interaction with C3PO, and produces large changes the cellular levels of microRNAs that have been implicated in heart disease. Here, we will test this novel connection between mechanical stress and gene regulation through caveolae / Gq/ PLC /C3PO. We will first determine the organization, concentration and oligomerization of Gq, its associated angiotensin receptor and PLC in caveolae domains using a series of fluorescence imaging and biochemical methods. We will then view their release from caveolae domains in real time as caveolae flatten upon the reversible application of osmotic pressure. We will determine the impact of osmotic stress on changes in the microRNA population due to PLC-C3PO association in real time using RNA beacons to monitor microRNA and fluorescence complementation to monitor protein association. We will test the ability of these microRNAs to promote physiological changes in the cell. Since we find that active siRNA processing affects Ca2+ through Gq, we will determine if the underlying mechanism involves changes in PLC-C3PO association that occur as the C3PO machinery is engaged.

 描述（由申请人提供）：肌肉细胞经历健康和不健康的适应性变化，以响应环境线索。一个值得注意的例子是心肌梗死后心肌细胞的肥大性生长。已知心脏肥大以某种方式通过G蛋白的G β q家族进行调节，G β q家族介导来自肥大剂如血管紧张素II的信号。G β q激活磷脂酶C-β（PLC β）酶以在细胞中产生Ca 2+信号。Rho/Rac蛋白参与长期适应性G蛋白应答。我们已经证明，G q的激活状态是稳定的小窝。小窝是膜内陷，其可以通过扁平化机械地适应细胞体积的增加。我们发现，这种扁平化释放了G q，并破坏了它与PLC q的关联。ca 2+信号 然后衰减。减少与G β q的结合允许PLC β q移动到胞质溶胶中以结合C3 PO。C3 PO通过促进RNA诱导的沉默复合物（RISC）的活性来有效地抑制基因的RNA沉默。我们已经发现，增加PLC β通过其与C3 PO的相互作用逆转特定基因的RNA沉默，并产生与心脏病有关的microRNA的细胞水平的巨大变化。在这里，我们将测试机械应力和基因调控之间的这种新的连接，通过小窝/ G q/ PLC/C3 PO。我们将首先使用一系列的荧光成像和生物化学方法来确定G β q、其相关的血管紧张素受体和PLC β在小窝结构域中的组织、浓度和寡聚化。然后，我们将在真实的时间内观察它们从小窝结构域中的释放，因为小窝在可逆的渗透压作用下变平。我们将使用RNA信标监测microRNA和荧光互补监测蛋白质缔合来确定渗透压对由于PLC β-C3 PO缔合而导致的microRNA群体变化的影响，这是真实的。我们将测试这些microRNA促进细胞生理变化的能力。由于我们发现活性siRNA加工通过G β q影响Ca 2+，我们将确定潜在机制是否涉及当C3 PO机制参与时发生的PLC β-C3 PO缔合的变化。