Novel Mechanisms Regulating G Protein and MAP Kinase Signaling

描述（由申请人提供）：G蛋白是介导细胞对各种信号（包括光、气味、味觉、激素和神经递质）的反应的关键分子开关。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生理反应和疾病预防至关重要。这项提案的目标是发现新的和根本上重要的机制G蛋白的调节，使用遗传上易处理的生物酵母作为模型系统。主要假设是G蛋白信号转导是通过G蛋白亚基的泛素化来调节的。该假说基于最近在酵母中的观察，即G β亚基（Gpa1）和G β亚基（Ste4）都是泛素化的，阻断G蛋白泛素化的突变体深刻地影响信号传导。提出了三个具体的目标：目标1：什么是G（Ste4泛素化的功能作用？抗遍在化的Ste 4截短突变体对信息素刺激高度敏感。我们的假设是，刺激依赖性泛素化的Ste4作为一种机制，下调信息素信号。为了测试这一点，我们将确定Ste4泛素化所需的关键残基，并产生特异性阻断Ste4泛素化的点突变体。然后，我们将确定Ste4泛素化的后果，通过详细比较信号转导，亚细胞定位，结合能力和野生型Ste4与Ste4的点突变体，阻止其泛素化的稳定性。 目标2：G（Ste4）是如何被泛素化的？Ste4泛素化在信息素刺激后被诱导。我们的假设是信息素诱导的Ste4磷酸化或其从G（Gpa1）解离触发其泛素化。为了验证这一点，我们将研究Ste4磷酸化位点突变体中的Ste4泛素化，缺乏负责Ste4磷酸化的激酶的突变体，以及缺乏G（Gpa1.或者，信息素刺激可以直接激活泛素化酶。为了测试这一点，我们将确定信息素治疗是否改变负责E2和E3酶在体外和体内的活性。 目的3：为什么Rsp5是激活G蛋白信号所必需的？破坏酵母中的E3连接酶Rsp5会严重减少G蛋白步骤的信号传导。Gpa1被Rsp5单泛素化，在核内体具有正信号传导作用。我们的假设是，Rsp5催化的单泛素化是将Gpa1靶向到内体以发出信号所必需的。将通过确定破坏Rsp 5对Gpa 1的内体定位和信号传导的影响来检验该假设。Rsp5催化的泛素化可能调节G蛋白活化的替代可能性也将被测试。 公共卫生相关性：G蛋白介导的信号通路的缺陷可导致多种疾病和紊乱，包括心脏病、高血压、失明和内分泌紊乱。该项目将揭示调节G蛋白信号传导的新机制，这将为理解疾病机制提供重要见解，并有助于开发新的治疗方法。

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.