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: 6873759
• 负责人: MARILYN Gist FARQUHAR
• 金额: $ 41.27万
• 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/6873759
• 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蛋白信号通路的组分。其中RGS蛋白不仅通过其RGS结构域作为G蛋白的GAP，而且还通过其独特的N-和C-末端与其他蛋白结合。我们已经集中在两种RGS蛋白，GAIP（RGS 19），一种用于Galpha 13的GAP，和RGS-PX 1，一种用于Galphas的GAP，其也具有PX结构域，将其鉴定为参与磷酸肌醇信号传导的SNX蛋白。我们已经获得的结果表明，RGS蛋白连接G蛋白信号传导到其他信号传导途径，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，如蛋白质降解。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组成部分，并建立它们的细胞定位，运输和功能。潜在的主题是，RGS蛋白质是GAP和连接信号事件的桥接分子，并且这些相互作用在空间上受细胞分布和运输的调节。我们将专注于三个具体目标：具体目标#1：评估RGS-PX 1在早期内体连接Galphas信号传导和EGF受体（EGFR）下调中的作用。这将通过评估激活β 2-肾上腺素能受体和Galphas对EGFR降解、MAP激酶激活、RGS-PX 1定位和Gd-GFP分布的影响来实现。我们假设RGS-PX 1用于将通过激活Galphas产生的信号与EGFR信号联系起来。具体目标#2：确定GIPC是否具有组织在生长因子信号传导和运输中起作用的信号传导复合物的功能。GIPC是一种PDZ蛋白，结合GAIP和TrkA生长因子受体。我们将使用去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Galpha 13-GAIP-GIPC-GIPN信号传导网络。我们将集中在这里i）分离的假定信号复合物含有GAIP，Galphai 3，和GPCR，ii）GIPN，一种新的GAIP相互作用的蛋白质，这是一个假定的E3连接酶的表征，和iii）使用生物信息学模型G？i-GAIP-GIPC/GIPN信令网络。这些研究有望大大扩展RGS蛋白在连接G蛋白和生长因子信号传导中的作用的知识。 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）中的广泛功能，RGS蛋白代表了用于开发药理学和抗肿瘤剂的有吸引力的靶标。