Regulation of Cardiac Adrenergic Function by S-nitrosylation

描述（由申请人提供）：越来越多的证据表明，一氧化氮（NO）通过多个功能相关的信号传导元件内的Cys残基的S-亚硝基化，调节激动剂诱导的脱敏和？肾上腺素能受体（？AR）。我们已经证明，NO脱氢酶和内源性S-亚硝基硫醇（SNO），S-亚硝基谷胱甘肽，保持心肌收缩力和防止下调？维持激动剂刺激期间的AR。NO/SNO对？可以在细胞系统中重现的AR信号传导和运输似乎在很大程度上由G蛋白偶联受体（GPCR）激酶GRK 2的S-亚硝基化介导。？-的其他组件AR系统也受到S-亚硝基化的调节，这表明NO/SNO通过靶向S-亚硝基化发挥广泛的功能作用。特别是，我们最近确定了？arrestins（？AR 1和？arr 2）作为目标？通过NO还原酶（eNOS和nNOS）的AR偶联的S-亚硝基化。S-亚硝基化？arr 2由eNOS在一个单一的关键位点（半胱氨酸410）调节其蛋白质-蛋白质相互作用与网格蛋白和衔接蛋白-2（AP-2）在体外和体内，从而促进激动剂介导的？2-AR内化。然而，差异化监管？AR 1和？arr 2的S-亚硝基化修饰以及这些修饰对心脏功能的影响尚未研究。我们的中心假设是，逮捕将提供一个基础，由没有控制？- AR贩运和信号传导，在健康和衰竭的心脏中具有重要影响。此外，我们预测，监管？arr 1-然后呢？通过S-亚硝基化的arr 2特异性相互作用将提供NO发挥其调节影响的主要机制。我们将实现以下具体目标：1.阐明激动剂依赖性S-亚硝基化的位点，细胞和组织中eNOS和nNOS抑制蛋白; 2.解释的后果？抑制S-亚硝基化AR内化和脱敏; 3.评估后果？-抑制S-亚硝基化AR依赖性信号传导;和4.评估以下人员的职能作用：在完整心脏中抑制S-亚硝基化。总的来说，这些研究应该提供基本的和新颖的见解？在健康和衰竭的心脏中，NO对AR的调节可能会开辟一个新的研究领域。公共卫生相关性：心力衰竭与两种减毒？肾上腺素能受体（？AR）信号和降低的一氧化氮（NO）生物利用度。因为NO通过S-亚硝基化作用来调节两者？AR致密化和下调，了解S-亚硝基化在？AR信号传导和贩运可能会提高对？AR功能障碍，并提出新的治疗方法，心力衰竭。

DESCRIPTION (provided by applicant): Accumulating evidence indicates that nitric oxide (NO), through S-nitrosylation of Cys residues within multiple, functionally interrelated signaling elements, regulates agonist-induced desensitization and internalization of ?- adrenergic receptors (?-ARs). We have demonstrated that NO synthases and the endogenous S-nitrosothiol (SNO), S-nitrosoglutathione, preserve cardiac contractility and prevent down-regulation of ?-ARs during maintained agonist stimulation. These effects of NO/SNO on ?-AR signaling and trafficking, which can be recapitulated in cellular systems, appear to be mediated, in significant part, by S-nitrosylation of the G protein- coupled receptor (GPCR) kinase, GRK2. Additional components of the ?-AR system are also regulated by S- nitrosylation, which suggests a broad functional role for NO/SNO, exerted through targeted S-nitrosylation. In particular, we have recently identified the ?-arrestins (?arr1 and ?arr2) as targets of ?-AR-coupled S- nitrosylation by NO synthases (eNOS and nNOS). S-nitrosylation of ?arr2 by eNOS at a single critical site (Cys 410) regulates its protein-protein interactions with clathrin and adapter protein-2 (AP-2) in vitro and in vivo, thereby promoting agonist-mediated ?2-AR internalization. However, differential regulation of ?arr1 and ?arr2 by S-nitrosylation and the consequences of these modifications for cardiac function have not been explored. Our central hypothesis is that S-nitrosylation of the ?-arrestins will provide a basis for control by NO of ?-AR trafficking and signaling, with important ramifications in healthy and failing hearts. Moreover, we predict that the regulation of ?arr1- and ?arr2-specific interactomes by S-nitrosylation will provide a principal mechanism through which NO exerts its regulatory influence. We will carry out the following specific aims: 1. Elucidate the sites of agonist-dependent S-nitrosylation of the ?-arrestins by eNOS and nNOS in cells and tissues; 2. Elucidate the consequences of ?-arrestin S-nitrosylation for ?-AR internalization and desensitization; 3. Assess the consequences of ?-arrestin S-nitrosylation for ?-AR-dependent signaling; and 4. Assess the functional roles of ?-arrestin S-nitrosylation in the intact heart. Collectively, these studies should provide fundamental and novel insights into ?-AR regulation by NO in both healthy and failing hearts and may open a new area of research. PUBLIC HEALTH RELEVANCE: Heart failure is associated with both attenuated ?-adrenergic receptor (?-AR) signaling and decreased nitric oxide (NO) bioavailability. Because NO acts through S-nitrosylation to regulate both ?-AR densitization and downregulation, understanding the mechanisms of action of S-nitrosylation in ?-AR signaling and trafficking may improve understanding of ?-AR dysfunction and suggest novel therapeutic approaches to heart failure.