CHARACTERIZATION AND REGULATION OF BETA-ENDORPHIN RECEPT

β-内啡肽受体的特征和调节

• 批准号: 2897683
• 负责人: NANCY M LEE
• 金额: $ 31.06万
• 依托单位: CALIFORNIA PACIFIC MED CTR RES INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2897683
• 关键词: affinity chromatography; animal tissue; antibody; antisense nucleic acid; binding proteins; cerebellum; clone cells; crosslink; dynorphins; endorphins; genetic library; genetically modified animals; high performance liquid chromatography; hybrid cells; laboratory mouse; ligands; neoplastic cell culture for noncancer research; neuropeptide receptor; neuropharmacology; opioid receptor; periaqueductal gray matter; protein purification; receptor binding; receptor coupling; receptor expression; transfection

Though opioid receptors were first identified almost 20 years ago, they still have not been purified. A major problem has been the difficulty in solubilizing them in a form which retains high ligand-binding activity and unaltered characteristics. One way around this problem is to covalently label the receptors prior to solubilization, for example, by cross-linking them to a peptide ligand such as beta-endorphin. Cross-linking technique has been successfully employed by several laboratories to identify polypeptides that bind beta-endorphin, but the twin problem of determining which these labelled bands represent bona fide receptors, and followed by the actual purification of these receptors remains. We propose to use a cross-linking technique to identify receptors for beta- endorphin, dynorphinA(1-13) and DADLE in several tissues, including rat periaqueductal gray (PAG), which is enriched in mu opioid receptors, NG108- 15 neuroblastoma x glioma hybrid cells, which contain a homogeneous population of delta receptors, and guinea pig cerebellum, which is enriched in kappa opioid receptors. To demonstrate the pharmacological relevance of cross-linked bands, we will apply several criteria, including selective competition by specific opioid ligands, coupling to G-proteins in the case of the PAG, and down-regulation by chronic agonist treatment in NG108-15 cells. Those bands that appear to be pharmacologically relevant will be purified using ligand-specific antibodies, such as anti-beta-endorphin-antibody, in conjunction with other procedures such as lectin affinity chromatography and HPLC. The purified proteins will be partially sequenced, oligonucleotides will be synthesized corresponding to these sequences, and used as probes to isolate cDNA from a rat brain library. We will further characterize the cross-linked bands by determining their levels in NG108-15 cell lines transfected with sense and antisense portions of the cDNA sequence that codes for OBCAM, an opioid binding protein purified from bovine brain. The antisense-transfected cells exhibit reduced 3H-diprenorphine binding, relative to non-transfected and sense- transfected NG108-15 cells; in contrast, muscarinic and alpha2-adrenergic binding in these cells do not differ from controls, suggesting the transfection has specifically affected opioid binding. We will also transfect COS-7 cells with OBCAM cDNA, and characterize these cells for opioid receptor binding, using both conventional receptor binding assays as well as cross-linking. Finally, we are in the process of creating a line of transgenic mice containing antisense OBCAM cDNA. Preliminary characterization of the first generation of mice suggests that their response to morphine may be altered. When breeding has continued through enough generations to establish a stable line of genetically altered mice, these animals will be thoroughly tested pharmacologically, including beta-endorphin cross-linking studies.

尽管阿片受体在近20年前首次被发现， 还没有被净化。 一个主要问题是， 以保持高配体结合活性的形式溶解它们， 不变的特征。 解决这个问题的一种方法是共价地 在溶解之前标记受体，例如通过交联 它们与肽配体（例如β-内啡肽）结合。 交联技术 已被多家实验室成功应用， 结合β-内啡肽的多肽，但确定 这些标记的条带代表真正的受体， 保留这些受体的实际纯化。 我们建议使用交联技术来识别β- 内啡肽，强啡肽A（1-13）和DADLE在几种组织，包括大鼠 中脑导水管周围灰质（PAG），其富含μ阿片受体，NG 108- 15神经母细胞瘤x胶质瘤杂交细胞，其中含有一个同质的 δ受体群体和豚鼠小脑，其富集 在κ阿片受体中。 证明以下药物的药理学相关性 交联带，我们将采用几个标准，包括选择性 通过特定的阿片样物质配体竞争，在这种情况下与G蛋白偶联， 以及NG 108 -15中慢性激动剂治疗的下调 细胞 将纯化那些看起来与DNA相关的条带 使用配体特异性抗体，例如抗β-内啡肽抗体， 结合其它方法如凝集素亲和层析 HPLC。 纯化的蛋白质将被部分测序， 将合成对应于这些序列的寡核苷酸，并且 用作探针从大鼠脑文库中分离cDNA。 我们将通过测定它们的分子量来进一步表征交联带。 用正义和反义部分转染的NG 108 -15细胞系中的水平 编码OBCAM的cDNA序列，OBCAM是一种阿片结合蛋白， 从牛脑中提纯的 反义转染的细胞表现出 相对于未转染和有义转染， 转染NG 108 -15细胞;相反，毒蕈碱和α 2-肾上腺素能 这些细胞中的结合与对照组没有差异，表明 转染特别影响阿片样物质结合。 我们还将 用OBCAM cDNA转染COS-7细胞，并对这些细胞进行表征， 阿片受体结合，使用常规受体结合测定法和 以及交联。 最后，我们正在创造一系列转基因小鼠， 含有反义OBCAM cDNA。 第一个的初步特征 这表明它们对吗啡的反应可能会改变。 当繁殖持续到足够的世代， 稳定的转基因小鼠品系，这些动物将被彻底 包括β-内啡肽交联研究。