MOLECULAR MECHANISM OF OPIOID RECEPTOR

阿片受体的分子机制

• 批准号: 6104003
• 负责人: HORACE LOH
• 金额: $ 13.43万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-15 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6104003
• 关键词: CHO cells; immunocytochemistry; immunoprecipitation; laboratory rabbit; molecular cloning; opioid receptor; phosphorylation; protein sequence; protein structure function; receptor binding; receptor expression; receptor sensitivity; stimulant /agonist; tissue /cell culture

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.

阿片类药物耐受和依赖的分子机制是一个课题 紧张的调查。 模型的开发是为了方便 了解这些阿片类药物的药理作用。 成神经细胞瘤 x 神经胶质瘤 NG108-15 细胞就是这样的模型之一。 以前的研究与此 模型表明，δ-阿片类药物的同质群体 该细胞系中的受体受到严格的细胞调节。 长期阿片受体激动剂治疗导致受体丧失 由于受体脱敏和受体下调而产生的反应。 至少在受体下调方面与其他观察结果平行 阿片受体类型是在长期给药的动物中获得的 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 宽容的问题。 之前的努力因缺乏 阿片受体试剂，例如受体特异性抗体。 现在与 最近克隆了 δ-阿片类药物，随后克隆了 mu-和 kappa-阿片类药物受体， 现在可以开发受体特异性抗体。 因此，在 目前的研究，我们建议开发针对 δ-阿片受体特异性 用根据以下方法合成的肽对兔子进行免疫接种来产生抗体 推导克隆的δ-阿片受体的一级序列，并进行免疫 兔子的受体蛋白在大肠杆菌中表达并纯化。 产生的抗体的特性将通过比较西方抗体来确定 分析从 CHO 细胞分离的膜，CHO 稳定转染 δ-阿片受体 cDNA 和 NG108-15 细胞。 免疫细胞化学将 用脑切片和这些抗体进行，以便利用 已知δ-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， 将建立免疫沉淀δ-阿片受体。 这 受体磷酸化作为受体机制的假设 将使用这些受体特异性来研究脱敏 抗体来检查受体的磷酸化状态 激动剂治疗。 δ-阿片受体将与其他受体分离 使用这些抗体的磷蛋白。 已知蛋白质的能力 将检查磷酸化δ-阿片受体的激酶。 程度 将通过肽检查磷酸化和磷酸化位点 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来研究 克隆的δ-阿片受体的分析。 δ-阿片受体克隆 将通过定点诱变或缺失诱变进行突变，其中 重点关注细胞质结构域中的丝氨酸和苏氨酸部分 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以下方式进行评估 COS7细胞瞬时表达和CHO细胞稳定转染 宽型和突变型 δ-阿片受体。 通过所有突变研究 和磷酸化研究，我们将确定磷酸化的作用 细胞对阿片类激动剂长期存在的适应。