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.

项目摘要 G蛋白信号传导（RGS）蛋白的调节剂在G蛋白偶联受体中起关键的调节作用（GPCR）信号传导。通过G蛋白依赖性和非依赖性机制，RGS蛋白质发挥作用在疾病中的重要作用，这引发了许多努力以药物为目标。这些但是，RGS蛋白被认为是困难的药物靶标，并确定了努力的努力。因此，控制RGS蛋白活性，表达和/或亚细胞定位的机制已成为一个重要的投资领域。我们的长期目标是确定RGS蛋白的水平和活动如何受监管，以确定监管网络中可吸毒的“软点”。我们的理由当前的建议是，识别调查RGS2的特定机制将发现治疗点干预以增加RGS蛋白水平和活性。 RGS2蛋白水平低或活性与多种病理，包括高血压，心力衰竭和哮喘，我们的中心假设是在药理上增强RGS2蛋白水平将具有广泛的临床意义。我们的目标提案是解解翻译后机制的表达，活性和亚细胞定位 RGS2。我们将重点关注调节RGS2蛋白水平和活性的两个至关重要但已理解的机制。蛋白酶体降解和磷酸化。我们确定了针对RGS2的Cullin-Ring E3连接酶（CRL）蛋白酶体降解，最近鉴定出RGS2中的DEGRON，仅F-box蛋白识别 44（FBXO44），CRL的底物识别成分。在AIM 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。我们还将确定是否FBXO44-RGS2是否结合由相关的CRL确定。 FBXO44仅在CUL4B/DDB1的上下文中降低RGS2，但是，细胞中的CUL1/SKP1复合物并不是表明FBXO44 RGS2特异性可能取决于 CRL。在AIM 2中，我们将确定磷酸化对RGS2功能的作用。磷酸化作用a 在信号转导级联中的中心作用，但是缺乏有关全球的全面信息 RGS2蛋白功能的磷酸化作用。我们以前的研究确定了对PKC的潜在重要性调节RGS2中的SRC激酶。我们将确定哪些结果是磷酸化的，以及 RGS2蛋白稳定性的后果，使用多种技术，包括Phostag凝胶电泳，体外激酶活性评估以及LC-MS。在AIM 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蛋白依赖性的影响和非依赖性RGS2在翻译的细胞，物理相关的细胞系和离体模型中的功能。这些研究的预期结果将是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，我们将针对RGS2的未来药物发现努力的重要侵害。