BIOCHEMICAL RESEARCH ON OPIOID RECEPTORS

阿片受体的生化研究

• 批准号: 2561669
• 负责人: Eric J Simon
• 金额: $ 10.59万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2561669
• 关键词: G protein; biological signal transduction; cytoskeleton; electrospray ionization mass spectrometry; intermolecular interaction; matrix assisted laser desorption ionization; opioid receptor; posttranslational modifications; protein purification; protein structure function; receptor binding; receptor expression; structural biology

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.

描述（申请人的摘要）： 这是高级科学家奖（K05）的申请。 这 申请人计划继续他长期的长期研究 阐明急性和慢性分子基础的术语目标 阿片类药物的影响。为此，我们正在研究内源性阿片类药物 系统和研究的重点是结构，功能和 调节不同类型的阿片类药物受体。 一种理解 该内源性神经肽/受体系统最终应具有 对治疗和预防药物滥用的重要意义 并改善疼痛的治疗。 第一个目的涉及活性MU阿片类药物的结构研究 我们已经纯化了受体蛋白（ORP）。 翻译后 纯化的MU受体的修饰，例如糖基化， 磷酸化和二硫键将由强大的 质谱技术与罗恩·比维斯（Ron Beavis）博士合作 这项技术的领先专家。 这种方法也将很有用 为了探索其他修饰，例如c-棕榈酰化 硫酰基试剂的末端半胱氨酸和半胱氨酸的烷基化。 早期制剂中存在的蛋白质会引起肽 在牛Mu阿片受体的序列中找不到我们 最近克隆的任何数据库都将被净化，并且 表征，因为它可能是MU的重要新型调节剂 受体功能。 半胱氨酸残基在受体功能中的作用将通过 位置定向的诱变和亲和力标记。 重点将开始 与配体结合的残留物以及C末端的残留物可能 被棕榈树。 脂肪酰化的作用将通过 比较非上酰胺化突变体的功能与野生的功能 类型受体并研究激动剂和拮抗剂的作用 以及其他有关动态棕榈酰化速率的受体调节剂 受体。 取代的半胱氨酸可及性法（SCAM）将 被用来探测阿片类药物的亲水接入缝隙 受体结合位点。 简而言之，结合位点或附近的氨基酸 将被半胱氨酸取代。 配体结合的灭活 亲水性SH试剂被视为半胱氨酸面对的证据 亲水通道裂缝。 第二个目的提出了探测阿片受体与 细胞骨架元素和连接受体的蛋白质的存在 到细胞骨架。 这种互动可能对正确至关重要 受体位置和方向，甚至针对 受体功能。在最终目标中，信号转导的各个方面 将研究，包括鉴定涉及的受体区域 在G蛋白激活中。