OPIOID CONTROL MECHANISMS OF SIGNAL TRANSDUCTION

描述（申请人摘要）： 本项目的长期目标是确定如何激活mu δ-阿片受体导致不同的细胞内信号。 尽管μ-和δ-阿片受体可能在细胞内传递信息， 类似的方式，滥用和依赖产生的明显区别， 阿片类药物的潜力表明， 发现了 对于这个项目，已经开发了一个细胞模型， 稳定转染的GH 3细胞中克隆的μ和δ阿片受体 与两种细胞内效应物腺苷酸环化酶和 Ca++通道。 这一独特而重要的模型将用于识别 μ-和δ-阿片信号转导之间的根本差异 瀑布 这将通过系统分析 阿片受体，G蛋白和效应器之间的相互作用， 独一无二的克隆人 首先，受体密度对μ-和 将确定与效应物的δ-偶联。 二、信号传导 在仅表达μ-阿片样物质、仅表达δ-阿片样物质或μ-阿片样物质和δ-阿片样物质两者的克隆中 将检查受体。 第三，阿片受体与 G蛋白将通过纯化激动剂刺激的 受体-G蛋白复合物。 第四，G蛋白的活化， 阿片受体将使用激动剂诱导的掺入来研究， [32 P]叠氮苯胺基-GTP转化为G-α亚基。 第五，组成 异源三聚体G蛋白（G-α、G-β和G-γ亚基） 负责将μ和δ受体与效应器偶联的是 通过使用靶向特异性G 蛋白质亚基 最后，Ca 2+通道与G alpha的关联 和/或G-β，γ亚基将在免疫沉淀后进行评估 G蛋白/Ca 2+通道复合物。 了解基本的差异， μ和δ阿片受体在细胞内传递信息的方式， 导致开发治疗阿片类药物滥用的新方法， 疼痛管理

DESCRIPTION (Applicant's Abstract): The long term objective of this project is to determine how activation of mu and delta-opioid receptors leads to distinct intracellular signals. Although mu- and delta-opioid receptors may relay intracellular messages in a similar fashion, a clear distinction in the abuse and dependence producing potential of opioids suggests significant differences are yet to be discovered. For this project, a cellular model has been developed in which cloned mu and delta-opioid receptors in stably transfected GH3 cells interact differently with two intracellular effectors, adenylyl cyclase and Ca++ channels. This unique and important model will be used to identify fundamental differences between mu- and delta-opioid signal transduction cascades. This will be accomplished by the systematic analysis of the interactions between opioid receptors, G proteins and effectors in these unique clones. First, the effect of receptor density on mu- and delta-coupling to effectors will be determined. Second, signal transduction in clones expressing only mu-, only delta, or both mu and delta-opioid receptors will be examined. Third, the association of opioid receptors with G proteins will be determined by purification of agonist-stimulated receptor-G protein complexes. Fourth, the activation of G proteins by opioid receptors will be studied using agonist-induced incorporation of [32P]azidoanilido-GTP into G-alpha subunits. Fifth, the composition of the heterotrimeric G proteins (G-alpha, G-beta- and G-gamma subunits) responsible for coupling mu and delta- receptors to effectors will be confirmed by the use of antisense oligonucleotides targeting specific G protein subunits. Finally, the association of Ca2+ channels with G alpha and/or G-beta,gamma subunits will be assessed after the immunoprecipitation of G protein/Ca2+ channel complexes. Understanding basic differences in the way mu and delta-opioid receptors relay information intracellularly could lead to the development of new methods for the treatment of opioid abuse and pain management.