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: 6738941
• 负责人: MARILYN Gist FARQUHAR
• 金额: $ 40.23万
• 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/6738941
• 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，我们确定了几种新的蛋白质，这些蛋白质是新型G蛋白信号通路的组成部分。其中包括RGS蛋白质，不仅通过其RGS结构域充当G蛋白的差异，而且还通过其独特的N-和C-terni结合了其他蛋白质。我们集中于两个RGS蛋白GAIP（RGS19），一个Galphai3的间隙和RGS-PX1，这是一个用于GALPHAS的差距，该差距还具有一个PX结构域，该域将其鉴定为参与磷酸固醇的SNX蛋白。我们获得了结果，表明RGS蛋白将G蛋白信号传导与其他信号通路联系起来，包括EGF和TRKA生长因子信号传导和磷酸肌醇信号，以及其他细胞过程，例如蛋白质降解。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成分，并确定其细胞位置，运输和功能。潜在的主题是RGS蛋白既是缝隙和桥接分子，它们连接信号事件，并且这些相互作用在空间上受细胞分布和运输的空间调节。我们将重点介绍三个特定目标：特定目标1：评估RGS-PX1在早期内体内链接GALLASE信号传导和EGF受体（EGFR）下调的作用。这将通过评估激活β2-肾上腺素能受体的影响以及对EGFR降解，MAP激酶激活，RGS-PX1定位以及Galphass-GFP的分布的效果。我们假设RGS-PX1用于通过激活GALPHANE与EGFR信号传导生成的信号。特定目的＃2：确定GIPC是否功能在生长因子信号传导和运输中组织起作用的信号复合物。 GIPC是一种PDZ蛋白，均结合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，一种蛋白质组学方法和RNAi实验来研究GIPC在信号传导和运输复合物组装中的作用。特定目的＃3：通过生化，形态学和生物信息学方法进一步定义Galphai3-Gaip-GIPC-GIPC GIPN信号网络。我们将集中在i）i）隔离含有GAIP，Galphai3和GPCR的假定信号传导复合物，ii）GIPN的表征，GIPN是一种新型的GAIP相互作用蛋白，是一种推定的E3连接酶，以及III），使用生物信息学用于G？i-Gaip-GIPC/GIPC/GIPC/GIPN信号网络模型。可以期望这些研究大大扩展对RGS蛋白在连接G蛋白和生长因子信号传导联系的知识。 由于它们在细胞功能的调节中的广泛功能，包括细胞生长，分化和有丝分裂的作用，RGS蛋白代表着有吸引力的药理学和抗肿瘤剂的靶标。