CHARACTERIZATION AND REGULATION OF BETA-ENDORPHIN RECEPT

尽管阿片受体是在近20年前首次发现的，但它们 仍然没有被提纯。一个主要的问题是难以在 以保留高配体结合活性的形式溶解它们，并 没有改变的特征。解决这个问题的一种方法是共价 在增溶之前标记受体，例如通过交联法 它们与多肽配体结合，如β-内啡肽。交联化技术 已经成功地被几个实验室用来识别 结合β-内啡肽的多肽，但确定的孪生问题 这些标记的条带代表真正的受体，然后是 这些受体的实际纯化情况仍然存在。 我们建议使用一种交联技术来识别β-受体。 包括大鼠在内的几种组织中的内啡肽、强啡肽A(1-13)和DADLE 中脑导水管周围灰质(PAG)，富含Mu阿片受体，NG108- 15神经母细胞瘤x胶质瘤杂交细胞，其中包含一个同源的 β受体和豚鼠小脑的数量，这是丰富的 在kappa阿片受体中。以证明其药理相关性 交叉链接带，我们将应用几个标准，包括选择性 与G蛋白偶联的特定阿片配体的竞争 在NG108-15中，PAG的表达和慢性激动剂治疗的下调 细胞。 那些看起来与药理相关的条带将被提纯 使用配体特异性抗体，如抗β-内啡肽抗体，在 结合其他程序，如凝集素亲和层析 和高效液相色谱仪。纯化的蛋白质将被部分测序， 将根据这些序列合成相应的寡核苷酸，以及 作为探针用于从大鼠脑文库中分离cDNA. 我们将通过确定它们的交联带进一步表征它们 正义和反义基因在NG108-15细胞中的表达 编码阿片结合蛋白OBCAM的cDNAs序列 从牛脑中提纯。反义基因转染的细胞表现出 与未转染组和正义组相比，~3H-异丙诺啡结合减少。 转染NG108-15细胞；相比之下，毒鼠强和α2-肾上腺素能 这些单元格中的绑定与控件没有区别，这表明 转染法对阿片类药物的结合有明显影响。我们还将 将OBCAM基因导入COS-7细胞，并对其进行鉴定 阿片受体结合，使用两种传统的受体结合分析作为 以及交联性。 最后，我们正在创造转基因小鼠的品系 含有反义OBCAM基因的表达载体。第一个的初步表征 小鼠的产生表明它们对吗啡的反应可能会发生变化。 当繁殖经过足够多的世代而建立起 稳定的转基因小鼠品系，这些动物将彻底 药理测试，包括β-内啡肽交联性研究。

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.