Novel Mechanisms Regulating G Protein and MAP Kinase Signaling

调节 G 蛋白和 MAP 激酶信号传导的新机制

• 批准号: 7882011
• 负责人: Yuqi Wang
• 金额: $ 22.13万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7882011
• 关键词: Affect; Animal Model; Binding; Biological Assay; Biological Models; Blindness; Cell Surface Receptors; Cell membrane; Defect; Development; Disease; Dissociation; Endocrine System Diseases; Endosomes; Enzymes; Event; Excision; Feedback; GTP-Binding Proteins; Genes; Goals; Hormones; Human; In Vitro; Lead; Light; Mediating; Membrane Proteins; Mitogen-Activated Protein Kinases; Modification; Molecular; Monitor; Mono-S; Neurotransmitters; Odors; Organism; Partner in relationship; Pathway interactions; Pheromone; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Post-Translational Protein Processing; Protein Subunits; Proteins; Public Health; Regulation; Research; Resistance; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Stimulus; Taste Perception; Testing; Ubiquitin; Ubiquitination; Yeasts; base; disorder prevention; experience; hypertensive heart disease; in vivo; insight; mutant; novel; novel therapeutics; protein activation; public health relevance; receptor; response; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): G proteins are key molecular switches that mediate cellular responses to a wide variety of signals including light, odor, taste, hormones, and neurotransmitters. Precise regulation of the intensity and duration of G protein-mediated signaling is critical for normal physiological responses as well as for disease prevention. The goal of this proposal is to uncover novel and fundamentally important mechanisms for G protein regulation, using the genetically tractable organism yeast as a model system. The main hypothesis is that G protein signaling is regulated via ubiquitination of G protein subunits. The hypothesis is based on recent observations in yeast that both G( subunit (Gpa1) and G( subunit (Ste4) are ubiquitinated, and mutants that block G protein ubiquitination profoundly affect signaling. Three specific aims are proposed: Aim 1: What is the functional role of G( Ste4 ubiquitination? A truncation mutant of Ste4 that is resistant to ubiquitination is hypersensitive to pheromone stimulation. Our hypothesis is that stimulus- dependent ubiquitination of Ste4 serves as a mechanism to down-regulate pheromone signaling. To test this, we will identify critical residues required for Ste4 ubiquitination and generate point-mutants that specifically block Ste4 ubiquitination. We will then determine the consequences of Ste4 ubiquitination by a detailed comparison of signaling, subcellular localization, binding ability and stability of wild type Ste4 versus point mutants of Ste4 that block its ubiquitination. Aim 2: How is G( Ste4 targeted for ubiquitination? Ste4 ubiquitination is induced upon pheromone stimulation. Our hypothesis is that pheromone-induced phosphorylation of Ste4 or its dissociation from the G( Gpa1 triggers its ubiquitination. To test this, we will examine Ste4 ubiquitination in Ste4 phosphorylation-sites mutants, mutants that lack kinase responsible for Ste4 phosphorylation, and mutants that lack the G( Gpa1. Alternatively, pheromone stimulation may directly activate the ubiquitinating enzymes. To test this, we will determine whether pheromone treatment alters activity of responsible E2 and E3 enzymes in vitro and in vivo. Aim 3: Why is Rsp5 required for the activation of G protein signaling? Disrupting E3 ligase Rsp5 in yeast severely diminishes signaling at the step of G protein. Gpa1 is mono-ubiquitinated by Rsp5 and has a positive signaling role at endosome. Our hypothesis is that Rsp5-catalyzed mono- ubiquitination is required for targeting Gpa1 to endosome to signal. This hypothesis will be tested by determining the effect of disrupting Rsp5 on endosomal localization and signaling of Gpa1. Alternative possibility that Rsp5-catalyzed ubiquitination may regulate G protein activation will also be tested. PUBLIC HEALTH RELEVANCE: Defects in G protein-mediated signaling pathways can lead to a variety of diseases and disorders including heart diseases, hypertension, blindness and endocrine disorders. This project will reveal novel mechanisms that regulate G protein signaling, which will provide significant insights for the understanding of disease mechanisms and aid in the development of new therapeutics.

描述（由申请人提供）：G蛋白是介导细胞对各种信号反应的关键分子开关，包括光、气味、味觉、激素和神经递质。精确调节G蛋白介导的信号传导的强度和持续时间对于正常的生理反应和疾病预防至关重要。本提案的目标是利用遗传易感生物酵母作为模型系统，揭示G蛋白调控的新颖且根本重要的机制。主要假设是G蛋白信号是通过G蛋白亚基的泛素化调控的。这一假设是基于最近在酵母中观察到的G（亚基）（Gpa1）和G（亚基）（Ste4）都泛素化，并且阻断G蛋白泛素化的突变体深刻地影响信号传导。提出了三个具体目标：目标1:G（Ste4）泛素化的功能作用是什么？抗泛素化的Ste4的截断突变体对信息素刺激敏感。我们的假设是刺激依赖性Ste4泛素化作为下调信息素信号传导的机制。为了验证这一点，我们将确定Ste4泛素化所需的关键残基，并生成特异性阻断Ste4泛素化的点突变体。然后，我们将通过详细比较野生型Ste4与阻断其泛素化的Ste4点突变体的信号传导、亚细胞定位、结合能力和稳定性来确定Ste4泛素化的后果。目标2:G（Ste4）如何靶向泛素化？Ste4泛素化是由信息素刺激引起的。我们的假设是信息素诱导的Ste4磷酸化或其与G（Gpa1）的分离触发了其泛素化。为了验证这一点，我们将检查Ste4磷酸化位点突变体、缺乏负责Ste4磷酸化的激酶的突变体和缺乏G（Gpa1）的突变体中的Ste4泛素化。或者，信息素刺激可能直接激活泛素化酶。为了验证这一点，我们将确定信息素治疗是否会在体内和体外改变E2和E3酶的活性。目的3：为什么Rsp5是G蛋白信号激活所必需的？破坏酵母E3连接酶Rsp5会严重减少G蛋白的信号传导。Gpa1被Rsp5单泛素化，在核内体中具有积极的信号作用。我们的假设是rsp5催化的单泛素化是靶向Gpa1到核内体传递信号所必需的。这一假设将通过确定破坏Rsp5对内体定位和Gpa1信号传导的影响来验证。rsp5催化的泛素化可能调节G蛋白活化的可能性也将被测试。