G-protein Coupled Neuropeptide Receptors in Ascaris suum

描述(申请人提供)：寄生线虫仍然是世界各地人和牲畜发病和死亡的主要原因。控制的负担主要落在化疗药物上，但令人担忧的是，对许多抗线虫药物的抗药性正在变得越来越普遍。对新药的需求十分迫切。FMRFamide样多肽是一类结构相关的小分子无脊椎动物神经肽，广泛存在于所有线虫的神经系统中。大多数FL对一系列线虫肌肉产生显著的强烈作用，在低至下午1点的浓度下引起深刻的收缩和松弛。它们的分布和生理效应表明，Flp对线虫的运动、取食和繁殖具有重要的控制作用。因此，它们的受体是有吸引力的新型药物靶点。近年来，在秀丽线虫体内发现了一些带有FLP配体的G蛋白偶联受体(GPCRs)。在这里，有人建议利用这一信息来鉴定寄生线虫猪蛔虫的FLP受体。该提案的具体目的是(1)检验与猪线虫FLP受体高度同源的GPCRs在猪链霉菌中表达的假说；(2)检验这些受体的配体是内源猪链霉菌FLP受体的假说。通过使用线虫序列的BLAST搜索从蛔虫EST数据库中鉴定出的候选猪链霉菌FLP受体将使用RACE PCR进行全面表征。这些受体将在一个异源系统中进行功能性表达，并用已知的猪链霉菌FLP进行筛选。FLP配体将使用FLEXStation II(分子设备)来确定，以监测与受体激活相关的细胞内钙水平的升高。这个为期两年的项目的成功完成将产生关于第一个寄生线虫神经肽受体的结构和功能信息。这将有助于进一步的实验，以便更好地了解线虫中这一重要的神经递质家族的生物学。它还将为药物开发研究提供一个底物。

DESCRIPTION (provided by applicant): Parasitic nematodes remain a major cause of morbidity and mortality to humans and livestock throughout the world. The burden of control falls mainly on chemotherapeutics but alarmingly, resistance to many anti-nematode drugs is becoming increasingly prevalent. There is pressing need for new drugs. FMRFamide-like peptides (FLPs) are a complex family of small, structurally-related invertebrate neuropeptides that are ubiquitous in the nervous systems of all nematodes. Most FLPs elicit remarkably potent effects on a range of nematode muscles, inducing profound contractions and relaxations at concentrations as low as 1 pM. Their distribution and physiological effects indicate FLPs are fundamental to the control of nematode locomotion, feeding and reproduction. Their receptors are therefore attractive novel drug targets. Recently, a number of G protein-coupled receptors (GPCRs) with FLP ligands have been identified in Caenorhabditis elegans. Here, it is proposed to use this information to identify FLP receptors in the parasitic nematode, Ascaris suum. The specific aims of the proposal are (1) to test the hypothesis that GPCRs with high homology to the C. elegans FLP receptors are expressed in A. suum and (2) to test the hypothesis that the ligands for these receptors are endogenous A. suum FLPs. Candidiate A. suum FLP receptors, identified from the Ascaris EST database through BLAST searches using the C. elegans sequences, will be fully characterized using RACE PCR. These receptors will be functionally expressed in a heterologous system and screened with known A. suum FLPs. FLP ligands will be identified using FLEXstation II (Molecular Devices) to monitor elevated intracellular calcium levels associated with receptor activation. Successful completion of this two-year project would produce structural and functional information on the first parasitic nematode neuropeptide receptor. It would facilitate further experiments to provide a better understanding of the biology of this important family of neurotransmitters in nematodes. It would also provide a substrate for drug development studies.