OPIOID CONTROL MECHANISMS OF SIGNAL TRANSDUCTION

描述(申请人摘要)： 该项目长期目标是确定如何激活MU 而增量阿片受体导致不同的细胞内信号。 尽管Mu和Delta阿片受体可能在脑内传递细胞内信息 类似的方式，在滥用和依赖产生上有明显的区别 阿片类药物的潜力表明，重大差异尚未显现 被发现了。对于这个项目，已经开发了一个细胞模型，其中 稳定转染的GH3细胞中Mu受体和Delta-阿片受体的克隆 与两种细胞内效应物的相互作用不同，腺苷环化酶和 Ca++通道。这一独特而重要的模式将用于识别 单位阿片类药物和增量类阿片类药物信号转导的根本区别 瀑布。这将通过对 脑内阿片受体、G蛋白和效应物的相互作用 独一无二的克隆。第一，受体密度对微核率的影响 将确定与效应器的增量耦合。第二，信号转导 在仅表达Mu-、仅表达Delta或同时表达Mu和Delta-阿片类药物的克隆中 将对受体进行检查。第三，阿片受体与 G蛋白将通过激动剂刺激的纯化来确定 受体-G蛋白复合体。第四，G蛋白的激活 阿片受体的研究将使用激动剂诱导的掺入 [32P]叠氮苯胺基-GTP转化为G-α亚基。第五，政府官员的组成 异源三聚体G蛋白(G-α、G-β和G-γ亚基) 负责将Mu和Delta受体偶联到效应器的将是 通过使用针对特定G的反义寡核苷酸确认 蛋白质亚基。最后，钙通道与Gα的联系 和/或G-β、γ亚基将在免疫沉淀后进行评估 G蛋白/钙离子通道复合体。了解中的基本差异 阿片受体和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.