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研究 阐明急性和慢性疾病的分子基础的长期目标 阿片类药物的影响。为此我们正在研究内源性阿片类药物 我们研究的重点是系统的结构、功能和 不同类型阿片受体的调节。 一种理解 这种内源性神经肽/受体系统最终应该具有 对药物滥用的治疗和预防具有重要意义 并改善疼痛的治疗。 第一个目标涉及活性 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， 磷酸化和二硫桥，将由强大的研究 与 Ron Beavis 博士合作的质谱技术， 该技术的领先专家。 这个方法也会很有用 探索其他修饰，例如 C- 的棕榈酰化 末端半胱氨酸和半胱氨酸通过巯基试剂的烷基化。 早期制剂中存在的蛋白质，可产生肽 在我们现有的牛 mu 阿片受体序列中未发现 最近克隆的，也不在任何数据库中，将被纯化并 特征，因为它可能是 mu 的重要的新型调节剂 受体功能。 将通过以下方式研究半胱氨酸残基在受体功能中的作用 定点诱变和亲和力标记。 重点将放在 参与配体结合的残基以及可能在 C 末端的残基 被棕榈酰化。 脂肪酰化的作用将通过以下方式进行检查 比较非棕榈酰化突变体与野生突变体的功能 类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 用于探测阿片类药物的亲水性进入缝隙 受体结合位点。 简而言之，结合位点内或附近的氨基酸 将被半胱氨酸取代。 配体结合失活 亲水性 SH 试剂被视为半胱氨酸面临 亲水进入裂缝。 第二个目标是探索阿片受体与 细胞骨架元件和连接受体的蛋白质的存在 到细胞骨架。 这种相互作用可能对于正确的 受体的位置和方向，甚至用于调节 受体功能。最终目标是信号转导方面 将进行研究，包括识别所涉及的受体区域 在G蛋白激活中。