OPIOID RECEPTOR MECHANISMS

阿片受体机制

• 批准号: 2517883
• 负责人: FEDOR MEDZIHRADSKY
• 金额: $ 17.79万
• 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/2517883
• 关键词: G protein; adenylate cyclase; antisense nucleic acid; biological signal transduction; calcium channel; cell cell interaction; clone cells; electroporation; fluidity; fluorescence spectrometry; guanosine triphosphate; laboratory rat; liposomes; membrane activity; membrane lipids; membrane reconstitution /synthesis; mixed tissue /cell culture; opioid receptor; phosphorylation; protein kinase A; receptor binding; receptor coupling; receptor expression; receptor sensitivity; tissue /cell culture

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.

该提案重点是阿片受体的分子机制 完整的神经细胞和质膜的脂质环境。 一个目标是确定阿片类药物信号转导的调节过程 在给定多种阿片类药物和非阿片类受体起作用的给定细胞中 同时共享换能器和效应蛋白（“交叉 - 说话”）。这项工作将表征那些过程或组成部分 受体信号传导将在一致的速率上限制 完整细胞的环境，并揭示受体适应的机制。 应用多种方法学方法（包括定量 配体 - 受体 - 转化器 - 效应器相互作用，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，酶测定，离子融合/重建 运输），选择性，化学计量和调节 MU和Delta阿片受体，可用于特定类型的腺苷酸环化酶和Ca2+ - 将评估通过G蛋白亚型的通道，包括角色 依赖CAMP的蛋白激酶。随后，相互调制 在两种阿片类药物和阿片类药物和抑制性肾上腺素能量之间 将在细胞中研究毒蕈碱受体系统 受体/换能器/效应器的浓度/可用性有选择性 改变以诱导适应。了解营养的意义 大脑中发生的因素和细胞对细胞通信发生 还将在神经元格利亚的条件下研究信号转导 体外相互作用以及受体功能的细胞中的相互作用 受差异化代理的影响。在表现出的神经细胞中 生化相关（营地）阿片类药物耐受性和依赖性， 受体和选择性G蛋白含量的调节以及 将评估耦合，G蛋白选择性的作用。在 连词，受体储备和受体-G的贡献 蛋白质偶联与激动剂的效力和效力在引起效应器中 将评估响应（腺苷酸环化酶，Ca2+运输）。在 总体上关注分子串扰对调节的关注，另一个目标是 表征有效调制o阿片类药物的机制 受体功能（构象，结合亲和力/容量，膜 动力学）由膜脂质和由生物物理特性 膜环境。使用脂质转移蛋白和融合技术 用于修饰膜，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 光漂白后的荧光和荧光恢复） 在作用上必不可少的脂质边界层围绕阿片类药物受体将 可以识别出血浆中受体和G蛋白的迁移率 确定的膜及其在阿片类信号调节中的作用 转导评估（碰撞耦合机制）。总而言之， 拟议的研究描述了使用高级方法的使用 生物化学，药理学和神经科学以阐明调节 分子，膜和细胞水平的阿片类药物功能。