MOLECULAR MECHANISM OF OPIOID RECEPTOR

阿片类药物耐受和依赖的分子机制是在 严密的调查。已经开发了模型以便于 了解这些阿片类药物的药理作用。神经母细胞瘤 X胶质瘤NG108-15细胞就是这样一个模型。以前对此的研究 模型表明，三角洲阿片类药物的同质种群 该细胞系中的受体处于严格的细胞调控之下。 慢性阿片类激动剂治疗导致受体丢失 受体脱敏和受体下调所致的反应。 至少在受体下调方面与其他 在长期给药的动物中获得了阿片受体类型 阿片受体选择性激动剂。因此，对分子的理解 受体脱敏和下调的基础可以阐明 容忍的问题。之前的努力因缺乏 阿片受体试剂，如受体特异性抗体。现在有了 最近克隆了β-阿片受体和mU-和kappa-阿片受体， 现在可以开发出受体特异性抗体。因此，在 目前的研究，我们建议发展到三角洲阿片受体特异性 用根据以下方法合成的多肽免疫兔的抗体 克隆的β-阿片受体一级序列的推导及免疫 在大肠杆菌中表达和纯化受体蛋白的兔。 产生的抗体的身份将通过比较西方 稳定转染CHO的CHO细胞膜的分离分析 Delta-阿片受体cDNA和NG108-15细胞。免疫细胞化学将 用脑片和这些抗体进行实验，以便利用 已知的增量-阿片受体分布来表征这些抗体。 这些抗体抑制阿片受体结合的能力， 免疫共沉淀法将建立β阿片受体。这个 受体磷酸化作为受体作用机制的假说 将使用这些受体特异性研究脱敏作用 检测受体磷酸化状态的抗体 激动剂治疗。Delta-阿片受体将与其他 使用这些抗体的磷蛋白。已知蛋白质的能力 本课程将研究磷酸化β-阿片受体的激酶。度度 多肽将检测磷酸化和磷酸化的部位。 免疫沉淀或免疫亲和纯化受体的图谱。 受体磷酸化的影响也将通过突变进行研究。 克隆的β-阿片受体的分析。Delta-阿片受体克隆 将通过定点突变或删除突变进行突变，具有 细胞胞浆结构域中丝氨酸和苏氨酸的研究 受体分子。这些突变对磷酸化受体的影响 脱敏和受体下调将通过 重组病毒在COS7细胞中的瞬时表达及在CHO细胞中的稳定表达 野生型和突变型β-阿片受体。通过所有的突变研究 和磷酸化研究，我们将确定磷酸化的作用 在细胞对阿片类激动剂长期存在的适应中。

Molecular mechanism of opioid tolerance and dependence is a subject under intense investigations. Models have been developed so as to facilitate the understanding of these opioid pharmacological effects. Neuroblastoma x glioma NG108-15 cells is one such model. Previous studies with this model have suggested that the homogeneous population of delta-opioid receptor in this cell line is under stringent cellular regulation. Chronic opioid agonist treatment resulted in a loss in receptor's responses due to receptor desensitization and receptor down-regulation. At least in receptor down-regulation parallel observations with other opioid receptor types were obtained in animals chronically administered opioid receptor selective agonists. Thus, understanding of molecular basis for receptor desensitization and down-regulation could illuminate the problem of tolerance. Previous efforts have been hampered by lack of opioid receptor reagents, such as receptor specific antibodies. Now with recent cloning of delta-opioid followed by mu- and kappa-opioid receptor, receptor specific antibodies could now be developed. Therefore, in current studies, we propose to develop to delta-opioid receptor specific antibodies by immunizing rabbits with peptides synthesized according to deduced primary sequence of cloned delta-opioid receptor, and immunizing rabbits with receptor proteins expressed in and purified from E. coli. Identities of antibodies produced will be established by comparing western analysis of membrane isolated from CHO cells, CHO stably transfected with delta-opioid receptor cDNAs, and NG108-15 cells. Immunocytochemistry will be performed with brain slices and these antibodies in order to utilize known delta-opioid receptor distribution to characterize these antibodies. The abilities of these antibodies to inhibit opioid receptor binding, to immunoprecipitate delta-opioid receptor will be established. The hypothesis of receptor phosphorylation as mechanisms for receptor desensitization will be investigated using these receptor specific antibodies to examine the phosphorylation states of receptor during agonist treatment. Delta-opioid receptor will be separated from other phosphoproteins using these antibodies. Abilities of known protein kinases to phosphorylate delta-opioid receptor will be examined. Degree phosphorylation and sites of phosphorylation will be examined by peptide mapping of immunoprecipitated or immunoaffinity purified receptors. Effect of receptor phosphorylation will be investigated also by mutation analysis of cloned delta-opioid receptor. Delta-opioid receptor clone will be mutated by site-directed mutagenesis or deletion mutagenesis, with the focus on serine and threonine moieties in the cytosolic domains of the receptor molecule. Effect of these mutations on phosphorylation, receptor desensitization and receptor down-regulation will be evaluated by transient expression in COS7 cells and stable transfection in CHO cells of wide type and mutant delta-opioid receptor. Through all mutation studies and phosphorylation studies, we will establish the role of phosphorylation in cellular adaptation to chronic presence of opioid agonists.