Spatial Regulation of RGS and G Protein Signaling

RGS 和 G 蛋白信号传导的空间调控

• 批准号: 7029556
• 负责人: MARILYN Gist FARQUHAR
• 金额: $ 5.43万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-17 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7029556
• 关键词: G protein; RNA interference; adrenergic receptor; biological signal transduction; epidermal growth factor; flow cytometry; fluorescence resonance energy transfer; growth factor receptors; immunoelectron microscopy; immunoprecipitation; laboratory mouse; laboratory rabbit; laboratory rat; mass spectrometry; mitogen activated protein kinase; protein localization; protein protein interaction; protein structure function; proteomics; tissue /cell culture

DESCRIPTION (provided by applicant): During the last five years, we identified several new proteins that are components of novel G protein signaling pathways. Among them are RGS proteins that not only serve as GAPs for G proteins through their RGS domains, but also bind to other proteins through their unique N- and C-termini. We have concentrated on two RGS proteins, GAIP (RGS19), a GAP for Galphai3, and RGS-PX1, a GAP for Galphas which also has a PX domain that identifies it as a SNX protein involved in phosphoinositide signaling. We have obtained results indicating that RGS proteins link G protein signaling to other signaling pathways, including EGF and TrkA growth factor signaling and phosphoinositide signaling, and to other cell processes such as protein degradation. The overall goal of the proposed work is to define the molecular components of the novel signaling networks we have uncovered and to establish their cellular location, trafficking and functions. The underlying themes are that RGS proteins are both GAPs and bridging molecules that link signaling events and that these interactions are spatially regulated by cellular distribution and trafficking. We will focus on three specific aims: Specific Aim #1: To assess the role of RGS-PX1 in linking Galphas signaling and EGF receptor (EGFR) down-regulation at early endosomes. This will be accomplished by assessing the effects of activating the beta2- adrenergic receptor and Galphas on EGFR degradation, MAP kinase activation, RGS-PX1 localization and, distribution of Galphas-GFP. We hypothesize that RGS-PX1 serves to link signals generated through activation of Galphas to EGFR signaling. Specific Aim #2: To determine if GIPC functions to organize signaling complexes that function in growth factor signaling and trafficking. GIPC, a PDZ protein, binds both GAIP and the TrkA growth factor receptor. We will use deconvolution and immunoelectron microscopy, a proteomics approach and RNAi experiments to investigate GIPC's role in assembly of signaling and trafficking complexes. Specific Aim #3: To further define Galphai3-GAIP-GIPC-GIPN signaling networks by biochemical, morphological and bioinformatics approaches. We will concentrate here on i) isolation of putative signaling complexes containing GAIP, Galphai3, and GPCR, ii) characterization of GIPN, a novel GAIP-interacting protein that is a putative E3 ligase, and iii) use of bioinformatics to model G?i-GAIP-GIPC/GIPN signaling networks. These studies can be expected to considerably expand knowledge of the role of RGS proteins in linking G protein and growth factor signaling. Due to their broad functions in regulation of cell functions, including cell growth, differentiation and mitogenesis, RGS proteins represent attractive targets for development of pharmacologic and anti-tumor agents.

描述（由申请人提供）：在过去的五年中，我们鉴定了几种新的蛋白质，它们是新型 G 蛋白信号传导途径的组成部分。其中包括 RGS 蛋白，它不仅通过其 RGS 结构域充当 G 蛋白的 GAP，而且还通过其独特的 N 端和 C 端与其他蛋白结合。我们集中研究了两种 RGS 蛋白：GAIP (RGS19)（Galphai3 的 GAP）和 RGS-PX1（Galphas 的 GAP），它也具有 PX 结构域，将其识别为参与磷酸肌醇信号传导的 SNX 蛋白。我们获得的结果表明，RGS 蛋白将 G 蛋白信号传导与其他信号传导途径（包括 EGF 和 TrkA 生长因子信号传导和磷酸肌醇信号传导）以及其他细胞过程（例如蛋白质降解）联系起来。拟议工作的总体目标是定义我们发现的新型信号网络的分子成分，并确定它们的细胞位置、运输和功能。基本主题是 RGS 蛋白既是连接信号事件的 GAP 又是桥接分子，并且这些相互作用在空间上受到细胞分布和运输的调节。我们将重点关注三个具体目标： 具体目标#1：评估 RGS-PX1 在早期内涵体中连接 Galphas 信号传导和 EGF 受体 (EGFR) 下调的作用。这将通过评估激活 β2-肾上腺素能受体和 Galphas 对 EGFR 降解、MAP 激酶激活、RGS-PX1 定位和 Galphas-GFP 分布的影响来实现。我们假设 RGS-PX1 有助于将 Galphas 激活产生的信号与 EGFR 信号传导联系起来。具体目标#2：确定 GIPC 是否能够组织在生长因子信号传导和运输中发挥作用的信号复合物。 GIPC 是一种 PDZ 蛋白，可结合 GAIP 和 TrkA 生长因子受体。我们将使用反卷积和免疫电子显微镜、蛋白质组学方法和 RNAi 实验来研究 GIPC 在信号传导和运输复合物组装中的作用。具体目标#3：通过生物化学、形态学和生物信息学方法进一步定义 Galphai3-GAIP-GIPC-GIPN 信号网络。我们将在这里集中讨论 i) 含有 GAIP、Galphai3 和 GPCR 的假定信号复合物的分离，ii) GIPN 的表征，GIPN 是一种新型 GAIP 相互作用蛋白，是假定的 E3 连接酶，以及 iii) 使用生物信息学来模拟 G?i -GAIP-GIPC/GIPN 信令网络。这些研究预计将大大扩展人们对 RGS 蛋白在连接 G 蛋白和生长因子信号传导方面的作用的认识。 由于其在调节细胞功能（包括细胞生长、分化和有丝分裂发生）方面的广泛功能，RGS 蛋白代表了药理学和抗肿瘤药物开发的有吸引力的靶标。