Spatial Regulation of RGS and G Protein Signaling

在过去的五年里，我们鉴定了几种新的蛋白质，它们是新的G蛋白的组成部分 信号通路。其中有RGS蛋白，它不仅是G蛋白的缺口，通过它们的 RGS结构域，但也通过其独特的N-末端和C-末端与其他蛋白质结合。我们一直专注于 两个RGS蛋白，GAIP(RGS19)，GO_i3的缺口，和RGS-PX1，GO_S的缺口，也有Px 它是一种参与肌醇磷脂信号转导的SNX蛋白。我们已经获得了 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 生长因子信号和肌醇磷脂信号，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定义新信号的分子组成！ 我们已经发现了网络，并确定了它们的蜂窝位置、贩运和功能。内幕消息 主题是RGS蛋白既是间隙分子又是连接信号事件的分子，这些 相互作用受ceHuJar分布和贩运的空间调节。我们将重点关注三个具体目标： 特定目的#1：评估RGS-PX1在调节GO_S信号和表皮生长因子受体中的作用 在早期内小体下调。这将通过评估激活_2-的影响来实现。 肾上腺素能受体和G(Zs)对EGFR降解、MAPK激活、RGS-PX1定位以及 GO_S-绿色荧光蛋白的分布。我们假设RGS-PX1用于连接通过激活i产生的信号 GO_STO EGFR信令。具体目标#2：确定GIPC是否起到组织信号复合体的作用 在生长因子信号转导和运输中的作用。GIPC是一种PDZ蛋白，与GAIP和TrkA Growth I结合 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 I 研究GIPC S在信号和运输复合体组装中的作用的实验。具体目标3：i 用生化、形态和生物信息学进一步确定Gczi3-GAIP-GIPC-GIPN信号网络 方法：我们将集中在i)含有GAIP，G(zi3，j)的假定信号复合体的分离 和GPCR，ii)GIPN的特性，GIPN是一种新的与GAIP相互作用的蛋白，可能是E3连接酶，以及III)USEL 以模拟GOD-GAIP-GIPC/GIPN信令网络。这些研究可以预期为 大大扩展关于RGS蛋白在连接G蛋白和生长因子信号中的作用的知识。好了！ 由于它们在调节细胞功能方面具有广泛的功能，包括细胞的生长、分化和有丝分裂。 RGS蛋白是开发药理和抗肿瘤药物的重要靶点。1

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 Go_i3, and RGS-PX1, a GAP for Go_s which also has a PX domain that identifies it as 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 signalin! networks we have uncovered and to establish their cellular location, trafficking and functions. The underlyin themes are that RGS proteins are both GAPs and bddging molecules that link signaling events and that these interactions are spatially regulated by ceHuJar distribution and trafficking. We will focus on three specific aims: Specific Aim #1: To assess the role of RGS-PX1 in tinking Go_s signaling and EGF receptor (EGFR) down-regulation at early endosomes. This will be accomplished by assessing the effects of activating the _2- adrenergic receptor and G(zs on EGFR degradation, MAP kinase activation, RGS-PX1 localization and, distribution of Go_s-GFP. We hypothesize that RGS-PX1 serves to link signals generated through activation of I Go_sto EGFR signaling. Specific Aim #2: To determine if GIPC functions to organize signaling complexes that I function in growth factor signaling and trafficking. GIPC, a PDZ protein, binds both GAIP and the TrkA growth I factor receptor. We wUi use deconvolution an(] immunoelectron microscopy, a proteomics approach and RNAi I experiments to investigate GIPC s role in assembiy of signaling and trafficking complexes. Specific Aim #3: I To further define Gczi3-GAIP-GIPC-GIPN signaling networks by biochemical, morphological and bioinformatics I approaches= We will concentrate here on i) isolation of putative signaling complexes containing GAIP, G(zi3,j and GPCR, ii) characterization of GIPN, a novel GAIP-interacting protein that is a putative E3 ligase, and iii) usel of bioinformatics to model God-GAIP-GIPC/GIPN signaling networks. These studies can be expected to I 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, t RGS proteins represent attractive targets for development of pharmacologic and anti-tumor agents. 1