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

G 蛋白信号转导 2 (RGS2) Regularo 的翻译后调节

基本信息

批准号：
10894543
负责人：
Benita Jenny Sjogren
金额：
$ 29.91万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10894543
关键词：
Area Arteries Asthma Basic Science Binding Binding Proteins Biochemical Biological Assay Biological Process CUL1 gene Cardiac Myocytes Cell Line Cells Clinical Complex Cullin Proteins DNA Damage Disease Drug Targeting Epigenetic Process Event F Box Domain Family Foundations Future G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins GTPase-Activating Proteins Genetic Transcription Goals Heart failure Heterotrimeric G Protein Subunit Hypertension In Vitro Intervention Intracellular Signaling Proteins Investigation Mass Spectrum Analysis Mediating Mission Modeling Molecular National Institute of General Medical Sciences Nature Outcome Study Pathology Phosphorylation Phosphorylation Site Phosphotransferases Physiological Play Post-Translational Regulation Prevention Protein Kinase C Proteins Public Health RGS Proteins Recombinants Regulation Research Resistance Role Signal Transduction Signaling Protein Smooth Muscle Myocytes Specificity Spottings Stimulus System Techniques Testing Therapeutic Therapeutic Intervention Transfection Ubiquitin-Proteasomal Pathway Up-Regulation Vascular Smooth Muscle constriction drug development drug discovery druggable target gel electrophoresis in vivo member novel pharmacologic phosphoproteomics protein activation protein degradation protein expression protein function protein protein interaction respiratory smooth muscle response small molecule src-Family Kinases therapeutic target ubiquitin-protein ligase

项目摘要

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.

项目概要 G 蛋白信号传导 (RGS) 蛋白的调节剂在 G 蛋白偶联受体中发挥关键调节作用 (GPCR) 信号传导通过 G 蛋白依赖性和非依赖性机制，RGS 蛋白发挥作用。在疾病中发挥着重要作用，这推动了许多针对其功能的药理学努力。然而，RGS 蛋白被认为是困难的药物靶点这一事实阻碍了努力，并且识别因此，控制 RGS 蛋白活性、表达和/或亚细胞定位的机制已成为我们的一个重要研究领域是确定 RGS 蛋白的水平和活性。监管，以识别监管网络内可药物的“软点”。目前的建议是，识别调节 RGS2 的具体机制将揭示治疗点增加 RGS 蛋白水平和活性的干预与低 RGS2 蛋白水平或活性相关。多种疾病，包括高血压、心力衰竭和哮喘，我们的中心假设是药理学上提高 RGS2 蛋白水平将具有广泛的临床意义。提议是破译调节表达、活性和亚细胞定位的翻译后机制我们将重点关注两个重要但尚未充分研究的调节 RGS2 蛋白水平和活性的机制：我们鉴定了一种针对 RGS2 的 Cullin-RING E3 连接酶 (CRL)。蛋白酶体降解，并且最近鉴定了 RGS2 中仅被 F-box 蛋白识别的降解决定子 44 (FBXO44)，CRL 的底物识别组件在目标 1 中，我们将使用生化和结构。我们还将确定 FBXO44-RGS2 是否与 FBXO44 相互作用。结合由相关的 CRL 决定，FBXO44 仅在 CUL4B/DDB1 的情况下降解 RGS2，但细胞中不是 CUL1/Skp1 复合物，表明 FBXO44 RGS2 特异性可能取决于在目标 2 中，我们将确定磷酸化对 RGS2 功能的作用。在信号转导级联中发挥核心作用，但是缺乏关于全球的全面信息我们之前的研究确定了磷酸化对 RGS2 蛋白功能的潜在重要性。我们将确定哪些残基被磷酸化，以及哪些残基被磷酸化。使用多种技术（包括 PhosTag 凝胶）对 RGS2 蛋白稳定性产生影响电泳、体外激酶活性测定以及 LC-MS 在目标 3 中，我们将确定功能。我们将确定翻译后 RGS2 调节改变的后果。 -独立的RGS2在转染细胞、生理相关细胞系和离体模型中发挥作用。这些研究的预期结果将是详细了解 RGS2 蛋白水平、活性和通过这些努力，我们将使得亚细胞定位受到翻译后机制的调节。未来针对 RGS2 的药物发现工作取得了重要进展。