2010/AFGN Collaborative Project: The Heterotrimeric G-Protein Interactome

拟南芥2010/AFGN合作项目：异三聚体G蛋白信号通路的相互作用网络。Jones，北卡罗来纳大学PI Joachim Ulhrig，科隆大学Co-PI Ralph Panstruga，马克斯普朗克植物育种研究所-科隆背景和科学目标/优点：拟南芥中的多种信号传导网络利用两种异源三聚体G蛋白复合物中的一种或两种来偶联由G蛋白偶联受体（GPCR）在细胞表面感知的信号，有一个七跨膜（7 TM）域，称为效应酶。 植物异源三聚体G蛋白复合物是示例性信号传导网络中的中心节点。 到目前为止，下游效应物和上游GPCR都没有明确确定。 这种信息的缺乏与人类和C。线虫G蛋白偶联途径，其中950个GPCR和十几个已知的效应物已被确定;因此，代表了后生动物中最广泛的信号网络。 没有后生动物GPCR和几乎没有典型的效应子在拟南芥基因组中编码，因此，需要无偏见的方法来填补这一知识空白。为了解决这个问题，描述了一种高通量相互作用筛选以鉴定与异源三聚体G蛋白复合物相互作用的蛋白质。 URL：其他信息和进度位于http://www.bio.unc.edu/Faculty/Jones/lab/2010Project/Gene设置：中央核心由以下基因定义：GPA 1（At 2g 26300）、AGB 1（At 4g 34460）、AGG 1（At 3g 63420）、AGG 2（At 3g 22945）和AtRGS 1（At 3g 26090）。目前正在更彻底地与具有这一核心、符合确定的优先标准的选定互动者进行合作。 对于那些选定的，反向和分子遗传学的方法来确定哪些分支的G蛋白信号网络这些元素的操作。 将通过插入诱变或RNAi技术敲除多达100个编码确认的相互作用物的基因，以确定与G蛋白突变体的已知表型相关的功能丧失表型。 将通过检查体内相互作用来深入研究较小的子集。测试相互作用物的体内相互作用，并将那些确认的相互作用物用作诱饵以启动另外几轮筛选。请注意，基因集随着进展而扩展。 基因功能：拟南芥G蛋白参与的生理过程已被表征。G蛋白相互作用物将在遗传上被放置在这些途径中的一个或多个中。 数据传播：数据将通过项目网站TAIR和互动数据库（如IntAct、DIP和bioGRID）公布。公共数据将包括筛选中的识别频率、筛选条件、基因表达模式、蛋白质类型、同源物、interlog以及与单个基因和其他基因特异性资源的链接。 这些数据将进行生物信息学和图论荟萃分析，以确定相互作用网络中的主要信号转导途径和蛋白质复合物（信号体）。与NSF 2010和AFGN计划的相关性：这项研究活动实质上有助于NSF的“基准基因功能”目标。这项研究的目的是确定G蛋白信号网络中完整的相互作用的元素集;因此，它满足了“探索典型网络和系统”的需要。“这个项目与另外两个2010年的项目协调工作，这些项目专注于拟南芥蛋白质的全球相互作用组。更广泛的影响：这项工作的更广泛的影响将通过本科生，预科生和博士后学生的培训来实现。 此外，哈佛大学和邵氏大学将在为期10周的暑期研究项目中共同指导3-6名少数民族学生。

Arabidopsis 2010/AFGN Collaborative Project: The Interaction Network for the Heterotrimeric G-Protein Signaling PathwayPI Alan M. Jones, University of North CarolinaPI Joachim Ulhrig, University of KolnCo-PI Ralph Panstruga, Max Planck Institute for Plant Breeding- KolnBackground and Scientific Goals/Merit: A variety of signaling networks in Arabidopsis utilize one or both of the two heterotrimeric G-protein complexes to couple signals that are perceived at the cell surface by G-protein coupled receptors (GPCRs), that have a seven-transmembrane spanning (7TM) domain, to enzymes called effectors. The plant heterotrimeric G protein complex is a central node in an exemplary signaling network. To date, neither the downstream effectors nor the upstream GPCRs have been unequivocally identified. This paucity of information stands in contrast to humans and C. elegans G protein-coupled pathways, where 950 GPCRs and more than a dozen known effectors have been identified; thus, representing the most extensive signaling network in metazoans. None of the metazoan GPCRs and almost none of the canonical effectors are encoded in the Arabidopsis genome; thus, non-biased approaches are needed to fill this knowledge gap. To address this problem, a high through-put interaction screen to identify proteins that interact with the heterotrimeric G protein complex is descirbed. URL: Additional information and progress is located at http://www.bio.unc.edu/Faculty/Jones/lab/2010Project/Gene Set: The central core is defined by the following genes: GPA1 (At2g26300), AGB1 (At4g34460), AGG1 (At3g63420), AGG2 (At3g22945), and AtRGS1 (At3g26090). Selected interactors with this core that meet the defined criteria for priority are being pursued more thoroughly. For those selected, reverse and molecular genetic approaches are applied to determine on what branches of the G-protein signaling network these elements operate. Up to 100 genes encoding confirmed interactors will be knocked out either by insertional mutagenesis or a RNAi technique in order to determine the loss of function phenotype with regard to known phenotypes of G-protein mutants. A smaller subset will be pursued in great depth by examining in vivo interactions. Interactors are tested for in vivo interaction and those confirmed interactors are used as bait to initiate additional rounds of screening. Note that the gene set expands as progress is made. Gene Function: The physiological processes involving Arabidopsis G protein have been characterized. G protein interactors will be placed genetically in one or more of these pathways. Data dissemination: Data will be made public through a project website, TAIR, and interaction data repositories such as IntAct, DIP and bioGRID. Public data will include frequency of identification in the screen, screen conditions, gene expression patterns, protein type, homologs, interlogs, and links to the individual genes and other gene-specific resources. The data will be subjected to bioinformatic and graph-theoretical meta-analyses to identify the main signal transduction pathways and protein complexes (signalosomes) in the interaction network. Key functional signal mediators (hubs) as well as abundant regulatory patterns will be identified.Relevance to the NSF 2010 and AFGN programs: This research activity substantially aids NSF's goal for "Benchmarking Gene Function." The research has as its aim to identify the complete interacting set of elements in the G-protein signaling network; thus, it meets the need for "exploring exemplary networks and systems." This project coordinates efforts with two other 2010 projects that are focusing on the global interactome of Arabidopsis proteins. Broader Impact: A broader impact of this work will be realized through the training of undergraduate, pre-, and post-doctoral students. In addition, UNC and Shaw University will jointly mentor 3-6 minority students in a 10-week summer research program.