Heterotrimeric G-protein regulation of neurotransmission in C. elegans

描述（由申请人提供）：通过异源三聚体gtpase （g蛋白）激活细胞效应酶和离子通道，调节细胞生长、有丝分裂和感觉知觉。g蛋白偶联受体是真核生物中最常用的信号转导器之一，是大约一半处方药的靶标。土壤线虫秀丽隐杆线虫（Caenorhabditis elegans）为研究异源三聚体g蛋白信号的保守性提供了许多实验优势。在胚胎中，G蛋白在不对称细胞分裂过程中调节控制有丝分裂纺锤体定位的微管力。在成人中，g蛋白通过改变突触活动和肌肉收缩的频率来协调动物的摄食、运动和其他行为。这些行为为研究细胞内和细胞间信号传递提供了方便和定量的方法。通过三条独立的实验线，我将研究通过两种g蛋白EGL-30 （Gctq）和GOA-1 （Ga0）信号传导的分子和细胞后果。活化的EGL-30与磷脂酰肌醇（4,5）二磷酸（PlPz）特异性磷脂酶C (p)家族成员EGL- 8相互作用。水解PIP？通过EGL-8释放第二信使肌醇1,4,5-三磷酸（IP3）和1,2-二酰基甘油（DAG）。相比之下，活化的GOA-1的直接效应仍然未知。为了找到这些，我将分离抑制超激活GOA-1信号表型的突变体。Koelle实验室的成员已经绘制了一个抑制因子的图谱，我将绘制其他抑制因子突变的图谱，确定编码因子如何调节信号传导，并测试它们是否作为GOA-1的直接效应器。其次，为了研究GOA-1如何拮抗EGL-30信号以调节突触活性，我将使用已建立的Ca+2荧光传感器和特定磷脂来可视化EGL-8在体内的活性。我将测试损害或刺激抑制性GOA-1信号传导的突变如何影响这些指标的行为和分布。这些原位生化实验将揭示遗传定义的信号通路的细胞生物学后果。最后，为了找到表达受信号调节的基因，我将通过微阵列分析比较goa-1和egl-30突变体的基因表达谱。这项研究将阐明神经系统如何控制肌肉收缩的频率。由于这些功能在人类大脑癫痫发作、肌肉震颤和神经退行性疾病（如帕金森病）中受到干扰，这些研究应该为靶向治疗提供合理的基础。

DESCRIPTION (provided by applicant): Signaling through heterotrimeric GTPases (G-proteins) activates cellular effector enzymes and ion channels to regulate cell growth, mitotic division, and sensory perception. G-protein coupled receptors are among the most commonly used signal transducers in eukaryotes and are the targets of about half of prescribed Pharmaceuticals. The soil nematode, Caenorhabditis elegans, offers many experimental advantages to investigate the conserved features of heterotrimeric G-protein signaling. In embryos, G- proteins regulate microtubule forces that control mitotic spindle positioning during asymmetric cell division. In adults, G-proteins coordinate animal feeding, motility, and other behaviors by altering synaptic activity and the frequency of muscle contractions. These behaviors offer convenient and quantitative assays to study intracellular and intercellular signaling genetically. Through three independent lines of experimentation, I will study the molecular and cellular consequences of signaling through two G-proteins, EGL-30 (Gctq) and GOA-1 (Ga0). Activated EGL-30 interacts with EGL- 8, the phosphatidylinositol (4,5) bisphosphate (PlPz)-specific Phospholipase C (p) family member. Hydrolysis of PIP? by EGL-8 releases the second messengers inositol 1,4,5-triphosphate (IP3) and 1,2- diacylglycerol (DAG). In contrast, direct effectors of activated GOA-1 remain unknown. To find these, I will isolate mutants that suppress hyperactivated GOA-1 signaling phenotypes. One suppressor has already been mapped by members of the Koelle lab, and I will map additional suppressor mutations, determine how the encoded factors regulate signaling, and test whether they act as direct effectors for GOA-1. Second, to study how GOA-1 antagonizes EGL-30 signaling to modulate synaptic activity, I will visualize EGL-8 activity in vivo using established fluorescent sensors of Ca+2 and specific phospholipids. I will test how mutations that impair or stimulate inhibitory GOA-1 signaling affect the behavior and distribution of these indicators. These in situ biochemical experiments will reveal the cell biological consequences of the signaling pathways defined genetically. Finally, to find genes whose expression is regulated by signaling, I will compare gene expression profiles in goa-1 and egl-30 mutants by microarray analysis. This research will shed light on how the nervous system controls the frequency of muscle contractions. As these functions are perturbed during human brain seizures, muscle tremors, and neurodegenerative diseases such as Parkinson's, these studies should inform a rational basis for targeted therapies.