OPIOID RECEPTOR MECHANISMS

阿片受体机制

• 批准号: 2116961
• 负责人: FEDOR MEDZIHRADSKY
• 金额: $ 16.4万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2116961
• 关键词: OPIOID; RECEPTOR; MECHANISMS

This proposal focuses on the molecular mechanisms of opioid receptors in intact neural cells and in the lipid environment of the plasma membrane. One goal is to identify regulatory processes of opioid signal transduction in a given cell in which multiple Opioid and non-opioid receptors function concurrently, sharing thereby transducer and effector proteins ("cross- talk"). This work will characterize those processes or components of receptor signaling that will become rate-limiting in the concerted environment of intact cells, and reveal mechanisms of receptor adaptation. Applying multiple methodological approaches (including quantitation of ligand-receptor-transducer-effector interactions, covalent protein modification, use of antibodies and antisense oligonucleotides, immunoblotting, receptor over-expression, cell membrane/liposome fusion/reconstitution with purified protein components, enzyme assays, ion transport), the selectivity, stoichiometry and regulation of coupling of mu and delta opioid receptors to type-specific adenylyl cyclase and Ca2+- channels through G protein subtypes will be assessed, including the role of cAMP-dependent protein kinase. Subsequently, the mutual modulation between the two opioid and an opioid and inhibitory adrenergic and muscarinic receptor systems will be studied in cells in which the concentration/availability of receptor/transducer/effector is selectively altered to induce adaptation. To understand the significance of trophic factors and of cell-to-cell communication occurring in brain, opioid signal transduction will also be studied under conditions of neuron-glia interaction in vitro, and in cells in which receptor function is influenced by differentiating agents. In neural cells exhibiting biochemical correlates (cAMP levels) of opioid tolerance and dependence, the regulation of receptor and of selective G protein content and coupling, and the role Of G protein selectivity will be evaluated. In conjunction, the contribution of receptor reserve and of receptor-G protein coupling to agonist potency and efficacy in eliciting an effector response (adenylyl cyclase, Ca2+ transport) will be assessed. Within the overall focus on regulation by molecular cross-talk, the other goal is to characterize the mechanisms underlying the potent modulation o opioid receptor function (conformation, binding affinity/capacity, membrane dynamics) by membrane lipids and by the biophysical property of the membrane environment. Using lipid transfer proteins and fusion techniques for membrane modification, fluorescent opioids and G proteins, and fluorescent techniques of high sensitivity and resolution (time-resolved fluorescence and fluorescence recovery after photobleaching), the functionally essential lipid boundary layer around opioid receptors will be identified, the mobility of receptor and G protein in the plasma membrane determined and its role in the regulation of opioid signal transduction assessed (collision-coupling mechanism). In summary, the proposed research describes the use of advanced approaches of biochemistry, pharmacology, and neuroscience to elucidate the regulation of opioid function at the molecular, membrane, and cellular level.

该提案重点关注阿片受体的分子机制 完整的神经细胞和质膜的脂质环境中。 目标之一是确定阿片类信号转导的调节过程 在多个阿片类和非阿片类受体发挥作用的给定细胞中 同时，共享转导蛋白和效应蛋白（“交叉 谈话”）。这项工作将描述这些过程或组件 受体信号传导将成为协调一致的速率限制 完整细胞的环境，并揭示受体适应机制。 应用多种方法（包括定量 配体-受体-转导子-效应子相互作用，共价蛋白 修饰、使用抗体和反义寡核苷酸， 免疫印迹、受体过表达、细胞膜/脂质体 与纯化的蛋白质成分融合/重建、酶测定、离子 运输），选择性，化学计量和耦合的调节 mu 和 delta 阿片受体针对类型特异性腺苷酸环化酶和 Ca2+- 将评估通过 G 蛋白亚型的通道，包括作用 cAMP 依赖性蛋白激酶。随后，互调制 两种阿片类药物和阿片类药物和抑制性肾上腺素能药物之间 毒蕈碱受体系统将在细胞中进行研究，其中 受体/转导器/效应器的浓度/可用性是选择性的 改变以诱导适应。了解营养的重要性 大脑中发生的因素和细胞间通讯，阿片类药物 信号转导也将在神经元-胶质细胞条件下进行研究 体外以及受体功能受限的细胞中的相互作用 受分化剂的影响。在神经细胞中表现出 阿片类药物耐受性和依赖性的生化相关性（cAMP 水平）， 受体和选择性 G 蛋白含量的调节 耦合，并且将评估 G 蛋白选择性的作用。在 结合，受体储备和受体-G 的贡献 蛋白质与激动剂的效力和诱导效应的功效的偶联 将评估反应（腺苷酸环化酶、Ca2+ 转运）。内 总体重点是分子串扰的调节，另一个目标是 描述阿片类药物有效调节的机制 受体功能（构象、结合亲和力/容量、膜 动力学）通过膜脂和生物物理性质 膜环境。使用脂质转移蛋白和融合技术 用于膜修饰、荧光阿片类药物和 G 蛋白，以及 高灵敏度和分辨率的荧光技术（时间分辨 荧光和光漂白后的荧光恢复）， 阿片受体周围功能必需的脂质边界层将 被鉴定，受体和G蛋白在血浆中的迁移率 膜的测定及其在阿片类信号调节中的作用 评估转导（碰撞耦合机制）。综上所述， 拟议的研究描述了先进方法的使用 生物化学、药理学和神经科学来阐明调节 在分子、膜和细胞水平上研究阿片类药物的功能。