OPIOID RECEPTOR MECHANISMS

本建议着重于阿片受体在脑内的分子机制。 在完整的神经细胞和质膜的脂质环境中。 一个目标是确定阿片类药物信号转导的调节过程 在多个阿片和非阿片受体起作用的给定细胞中 同时，由此共享转导蛋白和效应器蛋白(交叉 这项工作将描述这些过程或组件的特征 受体信号将成为协调的速率限制 完整细胞的环境，并揭示受体适应的机制。 采用多种方法学方法(包括量化 配体-受体-转导-效应器相互作用，共价蛋白质 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 与纯化蛋白质组分、酶分析、离子的融合/重组 运输)、耦合的选择性、化学计量和调节 Mu和Delta阿片受体对类型特异性腺酰环化酶和钙离子的作用 将评估通过G蛋白亚型的通道，包括其作用 CAMP依赖的蛋白激酶。随后，相互调制 在两种阿片类药物和一种阿片类药物之间以及抑制肾上腺素能和 将在细胞中研究M受体系统，在该细胞中 受体/转导/效应器的浓度/可用性是选择性的 改变以诱导适应。要理解营养的意义 脑内发生细胞间通讯的因素和阿片类药物 信号转导也将在神经元-胶质细胞的条件下进行研究。 在体外和受体功能的细胞内相互作用 受分化因子的影响。在神经细胞中表现出 阿片类药物耐受和依赖的生化相关性(cAMP水平)， 受体和选择性G蛋白含量的调节以及 偶联，并对G蛋白选择性的作用进行评估。在……里面 结合，受体储备和受体-G的贡献 蛋白质偶联与激动剂的效力和诱导效应器的效果 将评估反应(腺苷环化酶、钙转运)。在 总体上关注分子串扰的调节，另一个目标是 描述阿片类药物有效调制的潜在机制 受体功能(构象、结合亲和力/容量、膜 动力学)由膜脂和生物物理性质决定 膜环境。利用脂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 高灵敏度和高分辨率的荧光技术(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 经鉴定，血浆中受体和G蛋白的迁移率 膜的确定及其在阿片信号调节中的作用 转导评估(碰撞-耦合机制)。总而言之， 建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这一调节 在分子、膜和细胞水平上的阿片类药物功能。

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.