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

G 蛋白信号传导调节因子 2 (RGS2) 的翻译后调节

• 批准号: 10446200
• 负责人: Benita Jenny Sjogren
• 金额: $ 30.06万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10446200
• 关键词: Area; Arteries; Asthma; Basic Science; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Process; Blood Vessels; 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; Pharmacology; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Post-Translational Regulation; Prevention; Protein Kinase C; Proteins; Public Health; RGS Proteins; RGS2 gene; Recombinants; Regulation; Research; Resistance; Role; Signal Transduction; Smooth Muscle Myocytes; Specificity; Spottings; Stimulus; System; Techniques; Testing; Therapeutic; Therapeutic Intervention; Ubiquitin-Proteasomal Pathway; Up-Regulation; base; constriction; drug development; drug discovery; druggable target; gel electrophoresis; in vivo; member; nano; novel; phosphoproteomics; protein activation; protein degradation; protein expression; protein function; protein protein interaction; protein-tyrosine kinase c-src; respiratory smooth muscle; response; small molecule; src-Family Kinases; therapeutic target; ubiquitin-protein ligase

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蛋白信号调节蛋白（Regulator of G Protein Signaling，RGS）在G蛋白偶联受体（G Protein-coupled receptor，G-受体）中起着重要的调节作用 （GPCR）信号传导。通过G蛋白依赖性和非依赖性机制，RGS蛋白发挥作用。 在疾病中的重要作用，这促使许多努力来重新定位它们的功能。这些 然而，由于RGS蛋白被认为是困难的药物靶点， 因此，控制RGS蛋白活性、表达和/或亚细胞定位的机制已经变得 一个重要的调查领域。我们的长期目标是确定RGS蛋白的水平和活性是如何影响 监管，以确定在监管网络内的药物“软点”。我们的理由是， 目前的建议是，确定调节RGS 2的特定机制将揭示 干预以增加RGS蛋白水平和活性。低RGS 2蛋白水平或活性与 包括高血压、心力衰竭和哮喘，我们的中心假设是， 增强RGS 2蛋白水平的药物将具有广泛的临床意义。我们的目标是 我们的建议是破译翻译后机制调节表达，活性和亚细胞定位 的RGS 2。我们将集中在两个关键的，但研究不足，调节RGS 2蛋白水平和活性的机制; 蛋白酶体降解和磷酸化。我们鉴定了针对RGS 2的Cullin-RING E3连接酶（CRL）， 蛋白酶体降解，并且最近鉴定了RGS 2中仅由F-box蛋白识别的降解决定子 44（FBXO 44），CRL的底物识别组件。在目标1中，我们将使用生物化学和结构 方法来确定FBXO 44如何与RGS 2相互作用。我们还将确定FBXO 44-RGS 2是否 绑定由关联的CRL确定。FBXO 44仅在CUL 4 B/DDB 1的情况下降解RGS 2， 但在细胞中没有CUL 1/Skp 1复合物，这表明FBXO 44 RGS 2特异性可能取决于 的CRL。在目标2中，我们将确定磷酸化对RGS 2功能的作用。磷酸化作用 在信号转导级联中的核心作用，但是缺乏关于全球的全面信息。 磷酸化对RGS 2蛋白功能的作用。我们以前的研究确定了PKC的潜在重要性 和Src激酶在调节RGS 2中的作用。我们将确定哪些残基被磷酸化，以及 RGS 2蛋白稳定性的结果，使用广泛的技术，包括PhosTag凝胶 电泳、体外激酶活性测定以及LC-MS。在目标3中，我们将确定功能性 改变翻译后RGS 2调节的后果。我们将确定对G蛋白依赖性 和非依赖性RGS 2在转染细胞、生理相关细胞系和离体模型中均发挥功能。 这些研究的预期结果将是对RGS 2蛋白水平、活性和 亚细胞定位受翻译后机制调节。通过这些努力，我们将 这是未来针对RGS 2的药物发现工作的重要进展。