Novel Mechanisms Regulating the Heterotrimeric G Protein Complex

调节异源三聚体 G 蛋白复合物的新机制

• 批准号: 8987575
• 负责人: ALAN M. JONES
• 金额: $ 36.18万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8987575
• 关键词: Ablation; Affect; Agonist; Animal Model; Animals; Apoptosis; Arabidopsis; Arrestins; Behavior; Biological Models; C-terminal; Cell division; Cell surface; Cells; Clathrin; Coupled; Development; Discrimination; Elements; Endocytosis; Eukaryota; Evolution; Figs - dietary; G-Protein-Coupled Receptors; GTP Binding; GTP-Binding Protein Regulators; GTP-Binding Proteins; Genetic; Genetic Models; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Health; Heterotrimeric GTP-Binding Proteins; Hormones; Human; Human Engineering; Hydrolysis; In Vitro; Lead; Left; Ligands; Light; Molecular; Mouse-ear Cress; Organism; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiology; Plants; Protein Conformation; Protein Dephosphorylation; Proteins; RGS Proteins; Recruitment Activity; Recycling; Research; Resistance; Rest; Signal Pathway; Signal Transduction; Site; System; Tail; Testing; Thinking; Variant; Work; Yeasts; biological adaptation to stress; cell type; disorder control; in vivo; new therapeutic target; novel; pathogen; protein activation; protein complex; protein folding; receptor; response; tool

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.

描述（由申请人提供）：异源三聚体G蛋白偶联信号传导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。这种变异在生物体中遗传编码，进化了16亿年，代表了G蛋白信号系统的可塑性。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蛋白通过G蛋白偶联受体（GPCR）的激动剂刺激来激活以促进鸟嘌呤核苷酸交换，在拟南芥中，G蛋白自发地交换鸟嘌呤核苷酸而没有GPCR;相反，拟南芥利用G信号传导蛋白的7跨膜（受体样）调节剂（7 TM-RGS）的激动剂抑制来控制激活状态。动物细胞具有约800个GPCR来区分广谱信号（主要是激素激动剂），而拟南芥基本上具有一个由异源三聚体G蛋白和7 TM-RGS蛋白组成的G蛋白复合物。尽管如此，尽管有单一的G蛋白核心，遗传学证据表明，拟南芥G信号区分广谱的激动剂，就像动物细胞一样。该项目探讨了信号识别通过受体样激酶（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蛋白核心的物理组分。项目假设是：a）7 TM-RGS在其C末端尾部的配体依赖性磷酸化是G蛋白活化的关键步骤，B）未知的抑制蛋白折叠蛋白识别磷酸化的7 TM-RGS并募集网格蛋白以完成胞吞作用/G蛋白解偶联/活化，和c）磷酸化和胞吞的7 TM-RGS的再循环由未知的磷酸酶调节。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，因为它具有简单的异源三聚体G蛋白库，它为G信号传导提供了多细胞环境，它易于遗传操纵，并且它具有利用G信号传导的无数生理学，例如病原体抗性，胁迫反应，细胞分裂，光和光依赖性发育和程序性细胞死亡。具体而言，我们将：1）确定受体激酶的信息组与异源三聚体G蛋白复合物之间的物理关系，并确定同源配体如何改变物理组成和/或蛋白质构象。2)确定所选择的激酶组是否在体内和体外磷酸化7 TM-RGS蛋白（和其它G蛋白组分）以及这种磷酸化的细胞后果（例如7 TM-RGS内吞作用）。3)确定识别磷酸化AtRGS 1并控制其磷酸化状态的机制。这三个目标的成功实现将引入一个新的认识机制来调节G蛋白的激活。