Novel Mechanisms Regulating the Heterotrimeric G Protein Complex

描述(申请人提供)：异三聚体G蛋白偶联信号的核心元件在真核生物中是保守的，但调节G蛋白活性状态的机制不是。这种变异代表了G蛋白信号系统的可塑性，这种变异是在16亿年的进化过程中从基因上编码的生物。了解这种可塑性将揭示调节人类G信号的新方法。然而，在动物细胞中，G蛋白是通过激动剂刺激G蛋白偶联受体(GPCR)来促进鸟嘌呤核苷酸交换而激活的，而在拟南芥中，G蛋白自发地交换鸟嘌呤核苷酸而不是GPCR；相反，拟南芥利用激动剂抑制G信号蛋白的7个跨膜(受体样)调节器(7TM-RGS)来控制激活状态。动物细胞有大约800个GPCR来区分广泛的信号(主要是激素激动剂)，而拟南芥基本上只有一个G蛋白复合体，由异源三聚体G蛋白和7TM-RGS蛋白组成。尽管如此，尽管只有一个G蛋白核心，遗传证据表明，拟南芥G信号与动物细胞一样，可以区分广泛的激动剂。这个项目探索了信号识别是通过受体样激酶(RLK)实现的可能性。植物细胞编码约400个RLK，初步证据表明，一些RLK也是G蛋白核心的物理成分。该项目的假设是：a)配体依赖的，7TM-RGS在其C末端的磷酸化是G蛋白激活的关键步骤，b)未知的arrestin折叠蛋白识别磷酸化的7TM-RGS并招募clathrin完成内吞/G蛋白的解偶联/激活，c)磷酸化的7TM-RGS的循环，内吞的7TM-RGS受未知的磷酸酶调节。为了验证这些假设，我们将使用遗传模式生物拟南芥。拟南芥是阐明这一机制的理想系统，因为它有一个简单的异三聚体G蛋白谱系，它为G信号提供了一个多细胞环境，它易于遗传操作，它具有无数利用G信号的生理功能，如病原菌抗性、胁迫反应、细胞分裂、光和激素依赖的发育以及细胞程序性死亡。具体来说，我们将：1)确定一组信息丰富的受体激酶和异三聚体G蛋白复合体之间的物理关系，并确定同源配体如何改变物理组成和/或蛋白质构象。2)确定所选的一组激酶是否在体内和体外磷酸化7TM-RGS蛋白(和其他G蛋白成分)以及这种磷酸化的细胞后果(例如7TM-RGS内吞作用)。3)确定识别磷酸化AtRGS1的机制，并控制其磷酸化状态。这三个目标的成功完成将引入一种新认识的机制来调节G蛋白的激活。

DESCRIPTION (provided by applicant): The core elements of heterotrimeric G protein coupled signaling are conserved in eukaryotes but the mechanism to regulate the active state of the G protein is not. This variation, genetically encoded in organisms divergent by as much as 1.6 billion years of evolution, represents the plasticity of the G protein signaling system. Understanding this plasticity will reveal novel ways to regulate G signaling in humans. Whereas, in animal cells, the G protein is activated by agonist stimulation of a G-protein Coupled Receptor (GPCR) to promote guanine nucleotide exchange, in Arabidopsis, the G protein spontaneously exchanges guanine nucleotide without a GPCR; rather, Arabidopsis utilizes agonist inhibition of a 7 transmembrane (receptor like) Regulator of G Signaling protein (7TM-RGS) to control the activation state. Animal cells have ~800 GPCRs to discriminate among a broad spectrum of signals (mostly hormone agonists), in contrast to Arabidopsis which has essentially one G protein complex comprised of the heterotrimeric G protein and the 7TM-RGS protein. Nonetheless, despite the single G protein core, genetic evidence indicates that Arabidopsis G signaling discriminates a broad spectrum of agonists just as animal cells do. This project explores the possibility that signal discrimination is achieved by receptor-like kinases (RLK). Plant cells encode ~400 RLKs and preliminary evidence shows that some RLKs are also physical components of the G protein core. The project hypotheses are: a) ligand-dependent, phosphorylation of the 7TM-RGS at its C-terminal tail is the key step for G protein activation, b) an unknown arrestin-fold protein recognizes the phosphorylated 7TM-RGS and recruits clathrin to complete endocytosis/G protein uncoupling/activation, and c) the recycling of the phosphorylated, and endocytosed 7TM-RGS is regulated by an unknown phosphatase. To test these hypotheses, we will use the genetic model organism, Arabidopsis thaliana. Arabidopsis is the ideal system to elucidate this mechanism because it has a simple heterotrimeric G protein repertoire, it provides a multicellular context for G signaling, it is easily genetically manipulatd, and it has myriad physiologies that utilize G signaling, e.g. pathogen resistance, stress responses, cell division, light and hormone-dependent development and programmed cell death. Specifically, we will: 1) determine the physical relationship between an informative set of receptor kinases and the heterotrimeric G protein complex and determine how cognate ligands alter the physical composition and/or the protein conformations. 2) determine if the selected set of kinases phosphorylate the 7TM-RGS protein (and other G protein components) in vivo and in vitro and the cellular consequences of this phosphorylation (e.g. 7TM-RGS endocytosis). 3) Determine the mechanism to recognize phosphorylated AtRGS1 and to control its phosphorylation state. Successful completion of these three aims will introduce a newly-recognized mechanism to regulate G protein activation.