2010/AFGN Collaborative Project: The Interaction Network for the Heterotrimeric G-Protein Signaling Pathway

拟南芥中的多种信号网络利用异源三聚体g蛋白复合物将在细胞表面被g蛋白偶联受体（gpcr）感知的信号偶联到称为效应器的酶上。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，其中已鉴定出950个gpcr和十多个效应物，从而代表了后生动物中最广泛的信号网络。我们将使用高通量相互作用筛选来鉴定与异源三聚体G蛋白复合物相互作用的蛋白质。将对相互作用者进行体内相互作用测试，并将确认的候选药物用作启动额外筛选的诱饵。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）。选定的互动者将被更彻底地追求。反向遗传和分子生物学方法将被应用于确定这些元件在g蛋白信号网络的哪个分支上运作。通过插入突变或通过RNAi技术来确定功能丧失表型，多达100个编码已确认相互作用的基因将被敲除。一个较小的子集将通过检查相互作用和体内细胞定位进行深入研究。

A variety of signaling networks in Arabidopsis utilize heterotrimeric G-protein complexes to couple signals that are perceived at the cell surface by G-protein coupled receptors (GPCRs) to enzymes called effectors. To date, neither the downstream effectors nor the upstream GPCRs have been unequivocally identified. This paucity of information stands in contrast to human and C. elegans G protein-coupled pathways where 950 GPCRs and more than a dozen effectors have been identified thus representing the most extensive signaling network in metazoans. We will use a high through-put interaction screen to identify proteins that interact with the heterotrimeric G protein complex. Interactors will be tested for in vivo interaction and confirmed candidates will be used as bait to initiate additional rounds of screening. 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. Selected interactors will be pursued more thoroughly. Reverse genetic and molecular biological approaches will be applied to determine on which 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 via RNAi technique to determine the loss-of-function phenotype. A smaller subset will be pursued in great depth by examining interactions and cellular localization in vivo.