BIOCHEMICAL RESEARCH ON OPIOID RECEPTORS

描述（申请人摘要）： 这是一份高级科学家奖（K05）的申请书。 的 申请人计划继续他长期的研究， 长期目标是阐明急性和慢性 鸦片的影响。为此，我们正在研究内源性阿片样物质 系统，我们的研究重点是结构，功能， 调节不同类型的阿片受体。 的理解 这种内源性神经肽/受体系统最终应该有 对治疗和预防药物滥用的重要影响 改善了疼痛治疗。 第一个目标涉及活性μ阿片样物质的结构研究 受体蛋白（ORP）。 翻译后 纯化的μ受体的修饰，如糖基化， 磷酸化和二硫桥，将由强大的研究 与罗恩比维斯博士合作， 这项技术的顶尖专家 这种方法也将是有益的 用于探索其他修饰，例如C- 末端半胱氨酸和通过巯基试剂的半胱氨酸烷基化。 在早期制剂中存在的蛋白质，可产生缩氨酸 在牛μ阿片受体序列中没有发现， 最近克隆的，也不在任何数据库中，将被纯化， 其特征在于，因为它可能是一种重要的，新的μ调节剂， 受体功能 半胱氨酸残基在受体功能中的作用将通过 定点诱变和亲和标记。 重点将放在 参与配体结合的残基和C-末端中可能 被棕榈酰化。 脂肪酰化的作用将通过 比较非棕榈酰化突变体与野生型的功能 型受体，并通过研究激动剂和拮抗剂的作用 和其他受体调节剂对动态棕榈酰化速率的影响 受体。 替代半胱氨酸可及性方法（SCAM）将 用于探测阿片样物质的亲水性进入缝隙 受体结合位点 简言之，结合位点中或附近的氨基酸 将被半胱氨酸取代。 配体结合失活， 亲水性SH试剂被认为是半胱氨酸面对 亲水性通道裂缝。 第二个目标是探索阿片受体与 细胞骨架元素和连接受体的蛋白质的存在 到细胞骨架。 这样的相互作用对于正确的 受体的位置和方向，甚至调节 受体功能在最后的目标，信号转导方面， 将进行研究，包括识别涉及的受体区域 在G蛋白激活中。

DESCRIPTION (Applicant's Abstract): This is an application for a senior scientist award (K05). The applicant plans to continue his long standing research with the long term goal of elucidating the molecular basis of the acute and chronic effects of opiates. To this end we are studying the endogenous opioid system and the focus of our research is on the structure, function, and regulation of the different types of opioid receptors. An understanding of this endogenous neuropeptide/receptor system should ultimately have important implications for the treatment and prevention of drug abuse and improved treatment of pain. The first aim involves structural studies of the active mu opioid receptor protein (ORP) we have purified. Posttranslational modifications of the purified mu receptor, such as glycosylation, phosphorylation and disulfide bridges, will be studied by the powerful technique of mass spectrometry, in collaboration with Dr. Ron Beavis, a leading expert in this technology. This approach will also be useful for the exploration of other modifications such as palmitoylation of C- terminal cysteines and alkylation of cysteines by sulfhydryl reagents. The protein, present in earlier preparations, that gave rise to peptides not found in the sequence of the bovine mu opioid receptor we have recently cloned, nor in any database, will be purified and characterized, since it may be an important, novel regulator of mu receptor function. The role of cysteine residues in receptor function will be studied by site-directed mutagenesis and affinity labeling. The focus will be on residues involved in ligand binding and those in the C-terminal that may be palmitoylated. The role of fatty acylation will be examined by comparing the functions of non-palmitoylated mutants with those of wild type receptors and by studying the effects of agonists and antagonists and other receptor regulators on the dynamic palmitoylation rate of receptors. The Substituted Cysteine Accessibility Method (SCAM) will be employed to probe the hydrophilic access crevice of the opioid receptor binding site. Briefly, amino acids in or near the binding site will be replaced by cysteines. Inactivation of ligand binding by hydrophilic SH reagents is taken as evidence that the cysteine faces the hydrophilic access cleft. The second aim proposed probing the interaction of opioid receptors with cytoskeletal elements and the existence of proteins that link receptors to the cytoskeleton. Such an interaction may be essential for correct receptor location and orientation and even for the regulation of receptor functions. In the final aim, aspects of signal transduction will be studied, including identification of receptor regions involved in G protein activation.