Dissecting G Protein Pathways

描述（由申请人提供）： 异源三聚体G蛋白被广泛认为在以下方面发挥关键作用： 调节突触强度，但相对较少的是知道这些如何 信号通路调节突触囊泡胞吐和再循环。我们有 对两种G蛋白（Ga 0和Gaq） 调节线虫C.优雅我们已经表明 果阿-1 Ga 0和egl-30 Gaq拮抗乙酰胆碱（ACh） 释放C。线虫神经肌肉接头（NMJ）。我们发现了激动剂 激活每一条通路。5-羟色胺通过果阿-1 Ga 0起作用， 乙酰胆碱释放，而毒蕈碱激动剂，通过egl-30 Gaq，刺激乙酰胆碱释放， release.我们发现，这些G蛋白可能直接调节一些 突触囊泡回收方面，因为他们控制的丰度， 突触前突触融合蛋白结合蛋白（UNC-I3）在ACh释放位点的表达。我们 确定了两种蛋白质β-13和蛋白激酶C（kin 13 PKC）， 所需的佛波酯介导的刺激乙酰胆碱释放。我们提出 三个新的目标是确定这些G蛋白调节的机制， 神经递质释放 首先，我们将确定突触囊泡动力学的哪个方面受到调节 由果阿-1 Ga 0、egl-30 Gaq、gl-13和kin-13 PKC诱导。第二，我们将测试 Syntaxin complex的功能。第三，我们将确定 在何时何地kin-13 PKC调节ACh的释放。我们将测试 SNAP-25中潜在的kin-13 PKC磷酸化位点的功能重要性 -18。 总之，G蛋白对突触强度的调节与以下因素有关： 成瘾、情绪障碍、学习和记忆。考虑到强保守性 在这些跨越遗传学的途径中，我们的实验很可能会 为这些基本方面的机制提供新的见解 大脑中的信号传导。

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.