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。这个 申请人计划继续他与Long的长期研究 阐明急性和慢性精神分裂症分子基础的学期目标 鸦片类药物的作用。为此，我们正在研究内源性阿片类药物 系统，我们的研究重点是在结构，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/受体系统最终应该有 对治疗和预防药物滥用的重要影响 以及改善疼痛的治疗。 第一个目标是对活性阿片类药物进行结构研究。 我们已经纯化了受体蛋白(ORP)。翻译后 对纯化的Mu受体的修饰，如糖基化， 磷酸化和二硫键，将由强大的 质谱仪技术，与罗恩·比维斯博士合作， 这项技术的领先专家。这种方法也将是有用的 用于其他修饰的探索，如C-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。 一种蛋白质，存在于早期的制剂中，能产生多肽 在我们已有的牛MU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的，将被提纯并 特征的，因为它可能是一种重要的，新的Mu调节因子 受体功能。 半胱氨酸残基在受体功能中的作用将通过 定点突变和亲和标记。重点将放在 与配体结合有关的残基和可能存在于C末端的残基 是棕榈酰化的。脂肪酰化的作用将通过 非棕榈酰化突变体与野生突变体的功能比较 类型受体和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 感受器。替代半胱氨酸可及性方法(SCAM)将 被用来探测阿片类药物的亲水通道缝隙 受体结合部位。简而言之，结合部位中或其附近的氨基酸 将被半胱氨酸取代。通过以下方法失活配体结合 亲水性SH试剂被认为是半胱氨酸面对 亲水性通路裂隙。 第二个目标是探索阿片受体与 细胞骨架元件与连接受体的蛋白质的存在 到细胞骨架。这样的互动可能是正确的 受体的位置和方向，甚至调节 受体的功能。在最终目标中，信号转导的各个方面 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.