Dissecting G Protein Pathways

描述(由申请人提供)： 异三聚体G蛋白被广泛认为在 调节突触强度，但相对较少知道如何 信号通路调节突触囊泡的胞吐和再循环。我们有 对两种G蛋白(GA0和Gaq)进行了全面分析 调节线虫体内神经递质的释放。我们已经展示了 GoA-1GA0和EGL-30Gaq拮抗调节乙酰胆碱 在线虫神经肌肉接头(NMJ)释放。我们确定了激动剂 激活了这些通路中的每一个。5-羟色胺通过GoA-1 GA0发挥作用，抑制 ACh释放，而毒扁豆碱类激动剂通过EGL-30Gaq刺激ACh 放手。我们发现这些G蛋白可能直接调节某些 突触小泡循环的一个方面，因为它们控制着 ACh释放部位的突触前突触结合蛋白(UNC-L3)。而我们 确定了两种蛋白质UNC-13和蛋白激酶C(Kin13 PKC)，它们是 佛波酯介导的ACh释放刺激所必需的。我们建议 确定这些G蛋白调节机制的三个新目标 神经递质释放。 首先，我们将确定突触小泡动力学的哪个方面受到调控 GOA-1 GA0、EGL-30 GaQ、UNC-13和KIN-13 PKC。第二，我们将测试 UNC-13的功能重要性：语法蛋白复合体。第三，我们将确定 KIN-13PKC在何时何地调节ACh的释放。我们将测试 SNAP-25中潜在的KIN-13PKC磷酸化位点的功能重要性 和北卡罗来纳大学-18。 综上所述，G蛋白对突触强度的调节与 上瘾、情绪障碍、学习和记忆。考虑到强守恒性 在这些跨越系统发展史的途径中，我们的实验很可能将 对这些基本方面背后的机制提供了新的见解 大脑中的信号转导。

DESCRIPTION (provided by applicant): Heterotrimeric G proteins are widely thought to play a critical role in regulating synaptic strength, yet relatively little is known about how these signaling pathways regulate synaptic vesicle exocytosis and recycling. We have undertaken a comprehensive analysis of how two G proteins (Ga0 and Gaq) regulate neurotransmitter release in the nematode C. elegans. We have shown that goa-1 Ga0 and egl-30 Gaq antagonistically regulate acetylcholine (ACh) release at C. elegans neuromuscular junctions (NMJs). We identified agonists that activate each of these pathways. Serotonin, acting via goa-1 Ga0, inhibits ACh release whereas muscarinic agonists, acting via egl-30 Gaq, stimulate ACh release. We showed that these G proteins are likely to directly regulate some aspect of synaptic vesicle recycling, since they control the abundance of a pre-synaptic syntaxin binding protein (UNC-l3) at ACh release sites. And we identified two proteins UNC-13 and protein kinase C (kin13 PKC) that are required for phorbol ester-mediated stimulation of ACh release. We propose three new aims to determine the mechanisms by which these G proteins regulate neurotransmitter release. First, we will determine what aspect of synaptic vesicle dynamics is regulated by goa-1 Ga0, egl-30 Gaq, UNC-13, and kin-13 PKC. Second, we will test the functional importance of the UNC-13:Syntaxin complex. Third, we will determine where and when kin-13 PKC acts to regulate ACh release. And we will test the functional importance of potential kin-13 PKC phosphorylation sites in SNAP-25 and UNC-18. In summary, G protein regulation of synaptic strength has been implicated in addiction, mood disorders, learning, and memory. Given the strong conservation of these pathways across phylogeny, it is likely that our experiments will provide new insights into the mechanisms underlying these fundamental aspects of signal transduction in the brain.