Spatial Regulation of RGS and G Protein Signaling

描述（由申请人提供）：在过去的五年中，我们鉴定了几种作为新型G蛋白信号通路组分的新蛋白质。其中，RGS蛋白不仅通过其RGS结构域作为G蛋白的gap，还通过其独特的N-和c -末端与其他蛋白结合。我们集中研究了两种RGS蛋白，GAP (RGS19)， Galphai3的GAP，和RGS- px1， Galphas的GAP，也有一个PX结构域，将其识别为参与磷酸肌苷信号传导的SNX蛋白。我们已经获得的结果表明，RGS蛋白将G蛋白信号传导与其他信号传导途径联系起来，包括EGF和TrkA生长因子信号传导和磷酸肌苷信号传导，以及其他细胞过程，如蛋白质降解。这项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，并确定它们的细胞位置、运输和功能。潜在的主题是RGS蛋白是连接信号事件的间隙和桥接分子，这些相互作用受到细胞分布和运输的空间调节。我们将专注于三个具体目标：具体目标#1：评估RGS-PX1在早期内体连接Galphas信号和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-PX1定位和galphase - gfp分布的影响来完成。我们假设RGS-PX1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是否组织了在生长因子信号传导和运输中起作用的信号复合体。GIPC是一种PDZ蛋白，结合GAIP和TrkA生长因子受体。我们将使用反褶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：通过生化、形态学和生物信息学方法进一步定义galphai3 - gap - gipc - gipn信号网络。我们将集中在i)含有GAIP、Galphai3和GPCR的信号复合物的分离，ii) GIPN（一种新的GAIP相互作用蛋白，是一种假定的E3连接酶）的表征，以及iii)使用生物信息学来模拟G？i-GAIP-GIPC/GIPN信令网络。这些研究有望大大扩展对RGS蛋白在连接G蛋白和生长因子信号传导中的作用的认识。由于其在细胞功能调控方面的广泛功能，包括细胞生长、分化和有丝分裂，RGS蛋白是开发药物和抗肿瘤药物的有吸引力的靶点。

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.