DISSECTING G PROTEIN PATHWAYS

尽管异三聚体G蛋白占了很大一部分 哺乳动物大脑中的信号，许多第二信使和离子 受这些通路调控的通道还没有确定，很少 已知G蛋白通路之间的串扰，以及如何 这些信号决定了行为的变化。我们已进行了一项 G蛋白G/O信号转导机制的综合分析 线虫的神经系统。我们展示了GOA-1 (它编码G/O的阿尔法亚单位)在 通过5-羟色胺(5-羟色胺)发出信号。我们设计了行为分析方法 GOA-1和Gs发信号。使用这些检测方法，我们确定了13个 在基因下游起作用的神经调节缺陷基因 果阿1号和Gs下游的一处。我们已经克隆了其中的三个 下游基因。DGK-1和EGL-36位于GOA-1的下游，编码 二酰甘油激酶和Shaw型钾通道亚基， 分别进行了分析。Acy-1位于Gs下游，编码一种腺苷 循环酶。有趣的是，Acy-1突变也减少了GoA-1的信号转导。 1，暗示这些途径以某种方式相互作用。我们建议 行为、遗传、生化和生物物理实验 确定DGK-1、EGL-36和Acy-1在GOA-1信号转导中的作用。 首先，我们将确定GOA-1在哪里控制几个 行为。如果EGL-36、DGK-1和Acy-1以生化方式相互作用 GoA-1，它们必须在这些相同的细胞中表达。第二，我们将 确定GoA-1是否可以调节DGK-1的活性，以及Acy-1是否可以调节DGK-1的活性 1在转基因细胞系中表达。我们还将测试对DGK-1的依赖 通过测定反映GOA-1对磷脂酶C(PLC)的调节 线虫PLC的五种亚型中是否有任何一种是共表达的 GOA-1对共表达的PLC的调控作用 细胞。第三，我们将表征EGL-36通道的活性，其 与另外两个线虫Shaw亚基的相互作用，以及它是否 在转基因的组织培养细胞中受GoA-1调控。第四， 为了确定另一个潜在的Goa-1目标，我们将再克隆一个 下游基因mod(Nu62)。 综上所述，5-羟色胺与抑郁症、酗酒、 肥胖和自杀。考虑到对这些生物的强烈保护 我们的实验很可能会 确定药物治疗这些疾病的新靶点 严重的人类疾病。

Although heterotrimeric G proteins account for a large fraction of the signals in the mammalian brain, many of the second messengers and ion channels regulated by these pathways have not been identified, little is known about cross-talk between G protein pathways, and about how these signals determine changes in behavior. We have undertaken a comprehensive analysis of signaling by the G protein G/o in the nervous system of the nematode C. elegans. We showed that GOA-1 (which encodes the alpha subunit of G/o) plays an important role in signaling by serotonin (5-HT). We devised behavioral assays for signaling by GOA-1 and by Gs. Using these assays, we identified 13 mod (neuromodulation defective) genes that genetically act downstream of GOA-1 and one downstream of Gs. We have cloned three of these downstream genes. dgk-1 and egl-36 are downstream of GOA-1 and encode a diacylglycerol kinase and a Shaw type potassium channel subunit, respectively. acy-1 is downstream of Gs and encodes an adenylate cyclase. Interestingly, acy-1 mutations also reduce signaling by GOA- 1, implying that these pathways interact in some manner. We propose behavioral, genetic, biochemical, and biophysical experiments to determine what role DGK-1, EGL-36, and ACY-1 play in GOA-1 signaling. First, we will determine where GOA-1 acts to control several behaviors. If EGL-36, DGK-1, and ACY-1 biochemically interact with GOA-1, they must be expressed in these same cells. Second, we will determine whether GOA-1 can regulate the activities of DGK-1, and ACY- 1 in transfected cell lines. We will also test if dependence on DGK-1 reflects GOA-1 regulation of phospholipase C (PLC) by determining whether any of the five C. elegans isoforms of PLC are co-expressed with GOA-1, and if GOA-1 can regulate co-expressed PLCs in transfected cells. Third, we will characterize EGL-36 channel activity, its interactions with two other C. elegans Shaw subunits, and whether it is regulated by GOA-1 in transfected tissue culture cells. And fourth, to identify another potential GOA-1 target, we will clone one more downstream gene, mod(nu62). In summary, 5-HT has been implicated in depression, alcoholism, obesity, and suicidality. Given the strong conservation of these pathways across phylogeny, it is likely that our experiments will identify new targets for pharmaceuticals to treat these clinically important human disorders.