Characterization of the Endogenous Opioid and its Receptor in Tetrahymena: A Model System for the Evolution of ChemicalSignaling

四膜虫内源性阿片类药物及其受体的表征：化学信号进化的模型系统

• 批准号: 9105024
• 负责人: Fernando Renaud
• 金额: $ 42.17万
• 依托单位: University of Puerto Rico-Rio Piedras
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9105024&HistoricalAwards=false
• 关键词: Characterization; Endogenous; Opioid; its; Receptor

The long term goal of this project is to elucidate the nature of the signal transduction mechanisms in the protozoan ciliate Tetrahymena. This is an important question from the point of view of evolution because it seems likely that the mechanisms of signal transduction in metazoa originated from autocrine and paracrine mechanisms in unicellular eukaryotes. There is evidence that opioid mechanisms may play an important role in the modulation of phagocytosis in the protozoan ciliate Tetrahymena. In particular, a beta-endorphin-like peptide has been reported in this organism, and mammalian beta-endorphin inhibits phagocytosis in these cells with a high potency and efficacy. The proposed research will characterize two important elements of the opioid mechanism in this organism, the endogenous opioid and the receptor. This laboratory has confirmed by means of a radioimmunoassay the presence of a beta-endorphin-like peptide in Tetrahymena. The next goals are to establish the optimal conditions for the maximal expression of the peptide by studying how different factors, both environmental and biochemical, modulate the level of the peptide in the cell and in the culture medium, and then to purify the peptide from cell extracts. The effect of the purified peptide on phagocytosis in Tetrahymena and in metazoan models will be compared to that of mammalian beta-endorphin. The primary sequence of the purified peptide will be studied to determine its degree of homology with mammalian beta-endorphin or with other peptides that have been reported to have opioid-like activities. Studies on the putative beta-endorphin gene in tetrahymena will also be initiated, using standard recombinant DNA techniques. This will be done by probing a cDNA library with either a pro-opiomelanocortin (POMC) probe or an oligonucleotide probe based on the amino acid sequence. This will determine whether the putative beta-endorphin gene in Tetrahymena is coded independently or is part of a POMC gene, as in vertebrates. Studies of the opioid receptor will also be initiated to establish whether there is a correlation between the potency of opioid agonists and antagonists on phagocytosis, and their potency in radioligand displacement assays in membrane fractions. This work will also give information about the degree of opioid receptor microheterogeneity in Tetrahymena. In addition, studies will be done to determine how the properties of the receptor, including desensitization, are affected by changes in the ionic environment and by G-proteins. This will be done to compare the characteristics of the opioid receptor in Tetrahymena with other described subtypes. The results of this research will contribute to our understanding of the origins and mechanisms of chemical signaling. In particular, it will provide new information on the phylogenetic origin of beta-endorphin-like peptides and opioid receptors. These studies of the mechanisms by which such peptide mediators affect a ciliated protozoan may also shed new light on their mechanisms of action on the cells of higher animals.

该项目的长期目标是阐明原生动物纤毛虫四膜虫信号转导机制的性质。 从进化的角度来看，这是一个重要的问题，因为后生动物的信号转导机制似乎起源于单细胞真核生物的自分泌和旁分泌机制。 有证据表明阿片类机制可能在原生动物纤毛虫四膜虫吞噬作用的调节中发挥重要作用。 特别是，在该生物体中报道了β-内啡肽样肽，并且哺乳动物β-内啡肽以高效力和功效抑制这些细胞中的吞噬作用。 拟议的研究将表征该生物体中阿片类药物机制的两个重要要素，即内源性阿片类药物和受体。 本实验室通过放射免疫分析证实四膜虫中存在β-内啡肽样肽。 下一个目标是通过研究不同的环境和生化因素如何调节细胞和培养基中肽的水平，建立肽最大表达的最佳条件，然后从细胞提取物中纯化肽。 纯化的肽对四膜虫和后生动物模型中吞噬作用的影响将与哺乳动物 β-内啡肽的影响进行比较。 将研究纯化肽的一级序列，以确定其与哺乳动物β-内啡肽或已报道具有阿片样活性的其他肽的同源性程度。 还将利用标准重组 DNA 技术启动对四膜虫中假定的 β-内啡肽基因的研究。 这将通过使用阿片黑皮素原 (POMC) 探针或基于氨基酸序列的寡核苷酸探针探测 cDNA 文库来完成。 这将确定四膜虫中假定的 β-内啡肽基因是独立编码还是像脊椎动物中那样是 POMC 基因的一部分。 还将启动阿片受体的研究，以确定阿片激动剂和拮抗剂对吞噬作用的效力及其在膜组分中放射性配体置换测定中的效力之间是否存在相关性。 这项工作还将提供有关四膜虫阿片受体微观异质性程度的信息。 此外，还将进行研究以确定受体的特性（包括脱敏）如何受到离子环境和 G 蛋白变化的影响。 这样做是为了将四膜虫中阿片受体的特征与其他描述的亚型进行比较。 这项研究的结果将有助于我们了解化学信号的起源和机制。 特别是，它将提供有关β-内啡肽样肽和阿片受体的系统发育起源的新信息。 这些对这些肽介质影响纤毛原生动物的机制的研究也可能为它们对高等动物细胞的作用机制提供新的线索。