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

拟南芥2010/AFGN合作项目：异源三聚体g蛋白信号通路的相互作用网络/ / Alan M. Jones，北卡罗莱纳大学，Joachim Ulhrig, kolnuniversity - / / Ralph Panstruga, Max Planck Plant Breeding Institute - kolnmax Planck拟南芥中的多种信号网络利用两种异三聚体g蛋白复合物中的一种或两种，将具有7跨膜跨越（7TM）结构域的g蛋白偶联受体（gpcr）在细胞表面感知到的信号偶联到称为效应物的酶上。植物异三聚体G蛋白复合物是一个示例性信号网络的中心节点。到目前为止，下游效应体和上游gpcr都没有得到明确的鉴定。这种信息的缺乏与人类和秀丽隐杆线虫的G蛋白偶联途径形成对比，其中已经确定了950个gpcr和十多个已知的效应物；因此，代表了后生动物中最广泛的信号网络。在拟南芥基因组中没有后生动物gpcr和几乎没有典型效应物被编码；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，以识别与异源三聚体G蛋白复合物相互作用的蛋白质。Set：核心由以下基因定义：GPA1 (At2g26300), AGB1 (At4g34460), AGG1 (At3g63420), AGG2 （At3g22945）和AtRGS1 （At3g26090）。已选定的具有此核心且符合已定义的优先级标准的交互者将得到更彻底的追求。对于那些被选中的，反向和分子遗传学方法被应用于确定这些元件在g蛋白信号网络的哪个分支上起作用。通过插入突变或RNAi技术，多达100个编码已确认相互作用的基因将被敲除，以确定与已知表型的g蛋白突变体相关的功能表型损失。一个较小的子集将通过研究体内相互作用而深入研究。对相互作用物进行体内相互作用测试，这些被确认的相互作用物被用作启动额外筛选的诱饵。请注意，随着进展，基因集会扩大。基因功能：拟南芥G蛋白的生理过程已被表征。G蛋白相互作用物将被遗传地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和诸如完好无损、DIP和bioGRID等交互数据存储库公开。公共数据将包括筛选中鉴定的频率、筛选条件、基因表达模式、蛋白质类型、同源物、相互关联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形理论的meta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）。关键的功能信号介质（集线器）以及丰富的调节模式将被确定。与美国国家科学基金会2010和AFGN计划相关：这项研究活动极大地帮助了美国国家科学基金会“基准基因功能”的目标。该研究的目的是确定g蛋白信号网络中完整的相互作用元件集；因此，它满足了“探索示范性网络和系统”的需要。该项目与2010年另外两个关注拟南芥蛋白全球相互作用组的项目相协调。更广泛的影响：通过培养本科生、预科生和博士后来实现更广泛的影响。此外，北卡罗来纳大学和邵氏大学将共同指导3-6名少数民族学生进行为期10周的暑期研究项目。

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.