Spatial Regulation of RGS and G Protein Signaling

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，它们是新的G蛋白信号通路的组成部分。其中RGS蛋白不仅通过其RGS结构域作为G蛋白的间隙，而且还通过其独特的N-末端和C-末端与其他蛋白质结合。我们集中在两个RGS蛋白上，GAIP(RGS19)，Galphai3的缺口，以及RGS-PX1，GalpHas的缺口，它也有一个Px结构域，鉴定它是参与肌醇磷脂信号转导的SNx蛋白。我们已经获得的结果表明，RGS蛋白将G蛋白信号与其他信号通路联系起来，包括EGF和TrkA生长因子信号和磷脂酰肌醇信号，以及其他细胞过程，如蛋白质降解。这项拟议工作的总体目标是确定我们已发现的新型信令网络的分子组成，并确定它们的蜂窝位置、运输和功能。基本的主题是，RGS蛋白既是连接信号事件的间隙和桥梁分子，这些相互作用在空间上受到细胞分布和运输的调节。我们将集中于三个特定的目标：特定的目标1：评估RGS-PX1在连接Galphas信号和EGF受体(EGFR)在早期内吞体内下调中的作用。这将通过评估激活β2-肾上腺素能受体和GalpHas对EGFR降解、MAPK激活、RGS-PX1定位和Galpas-GFP分布的影响来实现。我们假设RGS-PX1用于将GalpHas激活产生的信号与EGFR信号联系起来。具体目标2：确定GIPC是否在组织在生长因子信号转导和贩运中起作用的信号复合体起作用。GIPC是一种PDZ蛋白，与GAIP和TrkA生长因子受体结合。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在信号和运输复合体组装中的作用。具体目标#3：通过生化、形态和生物信息学方法进一步定义Galphai3-GAIP-GIPC-GIPN信号网络。我们将集中在以下几个方面：1)分离含有GAIP、Galphai3和GPCR3的信号复合体；2)GIPN是一种新的与GAIP相互作用的蛋白，是一种可能的E3连接酶；3)利用生物信息学建立G？i-GAIP-GIPC/GIPN信号网络模型。这些研究有望极大地扩展RGS蛋白在连接G蛋白和生长因子信号中的作用。由于RGS蛋白具有广泛的细胞功能调节功能，包括细胞生长、分化和有丝分裂，因此是药物和抗肿瘤药物开发的诱人靶点。

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.