Heterotrimeric G alpha subunit activation by typical and atypical GEFs

描述(申请人提供)：我们治疗抑郁症、焦虑症和精神分裂症等疾病的能力极大地得益于我们对G蛋白信号网络的理解。已知异三聚体G蛋白信号调节多种生理过程，包括情绪。因此，G蛋白信号缺陷导致疾病也就不足为奇了。因此，我们对G蛋白信号的了解的进步直接导致了药物的开发，如三环类抗抑郁药/抗精神病药物和苯二氮卓类抗焦虑药物。然而，改进疗法的发展既需要发现新的药物靶点，也需要对这些新靶点如何调节信号有深入的了解。最近的研究发现了一类新的异三聚体G蛋白偶联系统的激活剂，然而，在我们对它们的作用机制和作用的细胞环境有更好的了解之前，这些蛋白质的治疗作用有限。在这项提案中，我的目标是表征Arr4，一种新的异源三聚体G蛋白信号的非受体激活剂。我最近发现Arr4与Gpa1相互作用，Gpa1是Gα-I的酵母同源物。此外，我发现Arr4可以作为Gpa1的真正的鸟嘌呤核苷酸交换因子，或称gef。在这里，我的目的是确定(I)典型和非典型Gβ-γ亚基在调节Arr4活性中的作用，(Ii)绘制Arr4和Gα亚基之间的结合面，(Iii)确定Arr4在细胞中作为环境基金的功能。为了达到这些目的，我将提纯涉及生化分析的各种蛋白质。此外，将使用标准的酵母遗传操作，以及一些已建立的体内和体外G蛋白激活测试。在整个提案中，我将描述酵母Arr4对酵母Gα，Gpa1的活性。由于这些蛋白质的酵母和人类同源物之间高度保守，我也将使用这些蛋白质的人类版本进行平行实验。酵母模型系统在提供信息方面有着悠久的历史，这些信息启发了哺乳动物系统中的类似发现。该提案的目的是确定Arr4等非受体GEF如何激活其同源G蛋白，并通过与小GTP酶的GPCRs和GEF进行比较，确定全球环境基金活动中的主题。这些信息将有助于我们更充分地利用GEF作为药物靶标。情绪障碍的治疗受益于针对G蛋白信号的药物。最近发现了一种新的蛋白质类来调节G蛋白信号，在这里我的目标是描述这类蛋白质的特征，以便创造出更好的治疗情绪障碍的药物。

DESCRIPTION (provided by applicant): Our ability to treat disorders such as depression, anxiety, and schizophrenia has benefited greatly from our understanding of G protein signaling networks. Heterotrimeric G protein signaling is known to regulate a wide variety of physiological process, including mood. Thus, it is not surprising that defects in G protein signaling lead to disease. Accordingly, advances in our understanding of G protein signaling have led directly to the development of drugs such as the Tri-Cyclic Anti-depressant/Anti-psychotics, and the Benzodiazepine family of anxiolytics. The development of improved therapeutics, however, will require both the discovery of new drug targets, and a refined understanding of how these new targets regulate signaling. Recent research has uncovered a new class of activators for heterotrimeric G protein coupled systems, however, these proteins are of limited therapeutic usefulness until we obtain a better understanding of their mechanism of action and the cellular context in which they function. In this proposal, I aim to characterize Arr4, a novel non-receptor activator of heterotrimeric G protein signaling. I recently identified that Arr4 interacts with Gpa1, a yeast homolog of G alpha-i. Furthermore, I found that Arr4 functions as a bona fide guanine-nucleotide exchange factor, or GEF, for Gpa1. Here, I aim to determine (i) the role of typical and atypical G beta-gamma subunits in regulating Arr4 activity, (ii) map the binding surface between Arr4 and G alpha subunits (iii) identify where in cells Arr4 functions as a GEF. To address these aims, I will purify the various proteins involved for biochemical analysis. Furthermore, standard genetic manipulation of yeast, as well as a number of established in vivo and in vitro assays for G protein activation will be used. Throughout this proposal, I will be characterizing the activity of yeast Arr4 on the yeast G alpha, Gpa1. Because of the high degree of conservation between the yeast and human homologues of these proteins, I will also conduct parallel experiments using the human versions of these proteins. The yeast model system has a long history of providing information that inspires analogous discoveries in mammalian systems. The goal of this proposal is to determine how non-receptor GEFs, such as Arr4, activate their cognate G proteins, and also to, by comparison to GPCRs and GEFs for small GTPases, identify themes in GEF activity. This information will aid in our ability to more completely utilize GEFs as drug targets. Treatment of mood disorders has benefited from drugs that target G protein signaling. A new class of proteins was recently found to regulate G protein signaling, and here I aim to characterize this class of proteins, in order to create better drugs to treat mood disorders.