Posttranslational regulation of Regulator of G protein Signaling 2 (RGS2)

项目摘要 G蛋白信号转导调节蛋白在G蛋白偶联受体中起着关键的调节作用 (GPCR)信令。RGS蛋白通过G蛋白依赖和非依赖两种机制发挥作用 在疾病中发挥重要作用，这推动了许多针对其功能的药理作用的努力。这些 然而，由于RGS蛋白被认为是困难的药物靶点，以及识别 因此，控制RGS蛋白活性、表达和/或亚细胞定位的机制已经成为 这是一个重要的调查领域。我们的长期目标是确定RGS蛋白的水平和活性 监管，以期找出监管网络内可用药的“软点”。我们的理论基础是 目前的建议是，确定调节RGS2的特定机制将揭示 干预以提高RGS蛋白水平和活性。低RGS2蛋白水平或活性与 包括高血压、心力衰竭和哮喘在内的一系列疾病，我们的中心假设是 从药物上提高RGS2蛋白水平将具有广泛的临床意义。我们的目标是 建议破译翻译后调节表达、活性和亚细胞定位的机制 RGS2。我们将集中在两个关键的，但未被充分研究，调节RGS2蛋白水平和活性的机制； 蛋白酶体降解和磷酸化。我们鉴定了一个针对RGS2的库林环E3连接酶(CRL)，用于 蛋白酶体降解，最近在RGS2中发现了仅由F-box蛋白识别的降解子 44(FBXO44)，CRL的底物识别成分。在目标1中，我们将使用生化和结构 确定FBXO44如何与RGS2相互作用的方法。我们还将确定FBXO44-RGS2 绑定由关联的CRL确定。FBXO44仅在CUL4B/DDB1的上下文中降级RGS2， 但在细胞中不存在CUL1/Skp1复合体，提示FBXO44 RGS2的特异性可能取决于 CRL。在目标2中，我们将确定RGS2功能的磷酸化作用。磷酸化起到了 在信号转导级联中的核心作用，但缺乏关于全球 磷酸化对RGS2蛋白功能的影响。我们之前的研究确定了PKC的潜在重要性 和Src激酶参与调节RGS2。我们将确定哪些残基是磷酸化的，以及 使用包括PhosTag凝胶在内的广泛技术对RGS2蛋白稳定性的影响 电泳法、体外激酶活性测定及LC-MS分析。在目标3中，我们将确定功能 翻译后RGS2调节改变的后果。我们将确定对G蛋白依赖的影响 在转基因细胞、生理相关细胞系和体外模型中，RGS2的功能都不依赖于AND。 这些研究的预期结果将是对RGS2蛋白水平、活性和 亚细胞定位受翻译后机制的调节。通过这些努力，我们将使 对未来针对RGS2的药物发现工作具有重要意义。

Project Summary Regulator of G protein Signaling (RGS) proteins play a key modulatory role in G Protein-coupled receptor (GPCR) signaling. Through both G protein-dependent and -independent mechanisms, RGS proteins play important roles in disease, and this has driven numerous efforts to pharmacologically target their function. These efforts are, however, hindered by the fact that RGS proteins are considered difficult drug targets, and identifying mechanisms that control RGS protein activity, expression and/or subcellular localization has therefore become an important area of investigation. Our long-term goal is to determine how levels and activity of RGS proteins is regulated, with a view of identifying druggable “soft-spots” within the regulatory network. Our rationale for the current proposal is that identification of specific mechanisms regulating RGS2 will uncover therapeutic points of intervention to increase RGS protein levels and activity. Low RGS2 protein levels or activity are associated with a wide range of pathologies, including hypertension, heart failure and asthma, and our central hypothesis is that pharmacologically enhancing RGS2 protein levels would have broad clinical implications. Our objective in this proposal is to decipher posttranslational mechanisms regulating expression, activity and subcellular localization of RGS2. We will focus on two crucial, but understudied, mechanisms regulating RGS2 protein levels and activity; proteasomal degradation and phosphorylation. We identified a Cullin-RING E3 ligase (CRL) targeting RGS2 for proteasomal degradation, and recently identified the degron in RGS2 that is recognized by F-box only protein 44 (FBXO44), the substrate-recognizing component of the CRL. In Aim 1 we will use biochemical and structural approaches to determine how FBXO44 interacts with RGS2. We will also determine whether FBXO44-RGS2 binding is determined by the associated CRL. FBXO44 degrades RGS2 only in the context of a CUL4B/DDB1, but not a CUL1/Skp1 complex in cells suggesting that FBXO44 RGS2 specificity may depend on the nature of the CRL. In Aim 2 we will determine the role of phosphorylation for RGS2 function. Phosphorylation plays a central role in signal transduction cascades, however there is a lack of comprehensive information on the global role of phosphorylation for RGS2 protein function. Our previous studies identified potential importance for PKC and Src kinase in regulating RGS2. We will determine which residues are phosphorylated, as well as the consequence for RGS2 protein stability, using a wide range of techniques, including PhosTag gel electrophoresis, in vitro kinase activity assays, as well as LC-MS. In Aim 3 we will determine the functional consequences of altered posttranslational RGS2 regulation. We will determine effects on G protein-dependent and -independent RGS2 functions in both transfected cells, physiologically relevant cell lines and ex vivo models. The expected outcome of these studies will be a detailed view of how RGS2 protein levels, activity and subcellular localization is regulated by posttranslational mechanisms. Through these efforts, we will make important inroads to future drug discovery efforts targeting RGS2.