OPIOID RECEPTOR MECHANISMS

该提案的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中。 目标之一是确定阿片信号转导的调节过程 在其中多种阿片样物质和非阿片样物质受体起作用的给定细胞中 同时，从而共享转导蛋白和效应蛋白（"交叉- talk "）。这项工作将描述这些进程或组成部分， 受体信号，这将成为限速的协调 完整细胞的环境，并揭示受体适应机制。 采用多种方法（包括量化 配体-受体-转导子-效应子相互作用，共价蛋白 修饰，抗体和反义寡核苷酸的使用， 免疫印迹，受体过表达，细胞膜/脂质体 与纯化蛋白质组分的融合/重构、酶测定、离子交换 运输），选择性，化学计量和耦合的调节， μ和δ阿片受体的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，相互调制 两种阿片类药物和一种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， 受体/换能器/效应器的浓度/可用性被选择性地 改变以诱导适应。为了理解营养的重要性， 因素和细胞间通讯发生在大脑，阿片类 信号转导也将在神经胶质细胞的条件下进行研究 在体外相互作用，并在细胞中，其中受体功能是 受差异化代理商的影响。在神经细胞中， 阿片类药物耐受性和依赖性的生化相关因素（cAMP水平）， 调节受体和选择性G蛋白含量， 偶联，和G蛋白选择性的作用将被评估。在 结合，受体储备和受体G的贡献 蛋白偶联激动剂的效力和引发效应物的功效 将评估反应（腺苷酸环化酶、Ca2+转运）。内 总体上集中于通过分子串扰进行调节，另一个目标是 描述了阿片类药物有效调节的机制 受体功能（构象、结合亲和力/能力、膜 动力学）的膜脂和生物物理性质的 膜环境利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）， 阿片受体周围的功能性必需脂质边界层将 受体和G蛋白在血浆中的迁移率 膜决定及其在阿片信号调节中的作用 评估转导（碰撞偶联机制）。总之， 拟议的研究描述了使用先进的方法， 生物化学、药理学和神经科学来阐明 阿片类药物在分子、细胞膜和细胞水平上的功能。

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.