Regulation of Heterotrimeric G proteins by non-receptor activators

总结/摘要： 异源三聚体G蛋白是细胞内主要的酶，其接收来自质膜G蛋白的信号， 蛋白偶联受体（GPCR），并将它们与广泛的细胞内效应物结合。我们的实验室 为研究G蛋白调控子Ric-8A和Ric-8B的功能做出了重要贡献。我们 Ric-8A和Ric-8B是G蛋白A亚基结合蛋白， 加快G β亚基释放GDP和结合GTP的速率。该活性类似于GPCR作用 G蛋白异源三聚体GPCR刺激G蛋白GDP/GTP交换，以激活 G蛋白传递信号我们还没有发现Ric-8蛋白促进G蛋白表达的确凿证据， 用于信令目的的GDP/GTP交换。我们确实发现小鼠中Ric-8A或Ric-8B基因的缺失 导致胚胎死亡Ric-8A或Ric-8B敲除胚胎干细胞的培养，所述胚胎干细胞从 死亡前的存活胚胎表明，细胞的G蛋白水平显著降低。这 促使我们研究Ric-8蛋白在调节G蛋白丰度中的作用。我们发现 Ric-8蛋白是分子伴侣，促进新产生的G β亚基的蛋白质折叠。当 G蛋白在缺乏Ric-8蛋白的细胞中产生，它们错误折叠并迅速降解。我们调和了 体外研究了具有折叠功能的Ric-8的GDP/GTP交换刺激活性， 蛋白质结合细胞中体外交换反应的中间产物，新合成的无核苷酸G 蛋白质，以促进第一次GTP结合。到目前为止，所有的证据都表明Ric-8蛋白质起作用， 组成性折叠G蛋白。这个新应用的前提是基于我们最近的数据，这些数据表明， Ric-8活性受到翻译后磷酸化的显著调节。我们将调查一个 导致Ric-8失调（去磷酸化）的促有丝分裂癌基因刺激与 可能是细胞G蛋白水平的重塑我们在长期的合作中取得了重大进展， 研究Ric-8蛋白调节G蛋白的结构基础。这项工作 应用将定义磷酸化调节Ric-8功能的方式。

Summary/Abstract: Heterotrimeric G proteins are the major intracellular enzymes that receive signals from plasma membrane G protein coupled receptors (GPCRs) and transduce them to a broad array of intracellular effectors. Our lab has contributed much to the understanding of the function of a pair of G protein regulators, Ric-8A and Ric-8B. We identified Ric-8A and Ric-8B as G protein  subunit binding proteins and showed that they possess the ability to accelerate the rate at which G subunits release GDP and bind GTP. This activity is similar to GPCR action towards G protein heterotrimers. GPCRs stimulate G protein GDP/GTP exchange for the purpose of activating the G proteins to transmit signals. We have yet to find conclusive evidence that Ric-8 proteins facilitate G GDP/GTP exchange for signaling purposes. We did find that deletion of the Ric-8A or Ric-8B genes in mice caused embryonic lethality. Culture of Ric-8A or Ric-8B knockout embryonic stem cells that were attained from viable embryos prior to death demonstrated that the cells had dramatically reduced levels of G proteins. This prompted our investigation into the role that Ric-8 proteins have in regulating G protein abundance. We found that Ric-8 proteins are molecular chaperones that facilitate protein folding of newly made G subunits. When G proteins are made in cells lacking Ric-8 proteins, they are misfolded and rapidly degraded. We reconcile the in vitro GDP/GTP exchange stimulatory activity of Ric-8 with the folding function by proposing that Ric-8 proteins bind the intermediate of the in vitro exchange reaction in cells, newly-synthesized, nucleotide-free G proteins to facilitate first time GTP binding. Until now, all evidence indicated that Ric-8 proteins acted constitutively to fold G proteins. The premise of this new application is based on our recent data that show that Ric-8 activities are subject to dramatic regulation by post-translational phosphorylation. We will investigate a new link between mitogenic oncogene stimulus that leads to Ric-8 deregulation (dephosphorylation) and possible remodeling of cellular G protein levels. We have made important strides in our long-time collaborative efforts to investigate the structural basis by which Ric-8 proteins regulate G proteins. The work in this application will define the way that phosphorylation regulates Ric-8 function.