CLONING OF CAPPA-OPIOID RECEPTOR VIA CDNA EXPRESSION

为了回答关于多种阿片类药物的众多问题之一， 受体，即这些受体是否代表不同的 基因产物，同一基因的不同剪接，或后 产品的跨国修改，这是目标， 目前的提议是克隆κ-阿片受体。 卡帕- 阿片受体将从豚鼠中克隆 小脑或人胎盘文库。 该文库将富集κ-阿片受体克隆， 3组探针：（a）μ-和 δ-阿片受体克隆;（B）合成的扣除探针 从含有低水平kappa- 含有高水平κ-阿片样物质的阿片样物质受体 受体，即慢性κ激动剂（U50 - 488）或拮抗剂 （MR2266）治疗将用于改变κ阿片受体 水平和因此mRNA水平;和（c）寡脱氧核苷酸 的推定跨膜区V、VI和VII的序列， 克隆的8-肾上腺素能受体。 杂交的cDNA克隆 使用前两组探针或使用所有三组探针 将在先前缺乏阿片样物质的真核生物中表达 受体活性。 诱导κ-阿片受体的克隆 结合活性将被亚克隆到Sp6载体中用于有义和 反义RNA合成。 如此合成的RNA将被 注射到青蛙卵母细胞中， 调节Ca +2通道将被用来证实 克隆人的身份 将开发针对 推导的肽序列，并将用于免疫沉淀 配体-κ-阿片受体复合物和/或用于抑制 脑细胞膜中的κ-阿片受体结合活性。 等 抗体也将用于免疫细胞化学分析， κ-阿片受体分布，并与文献报道的 推定的κ结合位点的分布。 的身份 κ-阿片受体克隆将进一步得到证实， 125I-B-内啡肽-受体复合物的分离和测序 当在μ-和δ-存在下进行标记时， 阿片配体。 的基因结构和核苷酸序列 将确定并比较κ-阿片样物质受体克隆 与μ-和δ-阿片受体克隆的相似。 缺失和 将进行核苷酸插入突变研究， 研究κ-阿片受体的结构要求 活动

In order to answer one of the many questions on the multiple opioid receptors, i.e. whether or not these receptors represent different gene products, different splicing of the same gene, or post- transnational modification of the products, it is the goal of the current proposal to clone for the kappa-opioid receptor. Kappa- opioid receptor will be cloned from either the guinea pig cerebellum or human placenta library by the cDNA expression method. The library will be enriched in kappa-opioid receptor clones with 3 sets of probes: (a) restriction enzyme fragments of the mu- and delta-opioid receptor clones; (b) subtraction probes synthesized from hybridizing mRNAs from tissues containing low level of kappa- opioid receptor from that containing high level of kappa-opioid receptor, i.e. chronic kappa agonist (U50-488) or antagonist (MR2266) treatment will be used to alter the kappa-opioid receptor level and hence mRNA levels; and (c) the oligodeoxynucleotides sequences of the putative transmembrane regions V, VI and VII of the cloned 8-adrenergic receptor. The cDNA clones which hybridize with the first two sets of probes or with all three sets of probes will be expressed in eukaryotes previously devoid of opioid receptor activities. Clones which induced kappa-opioid receptor binding activity will be subcloned into Sp6 vectors for sense and anti-sense RNA synthesis. The RNAs thus synthesized will be injected into frog oocytes and the ability of kappa agonist to regulate the Ca+2 channels will be used to substantiate the identity of the clones. Antibodies will be developed against the deduced peptide sequence and will be used to immunoprecipitate ligand-kappa-opioid receptor complex and/or used to inhibit the kappa-opioid receptor binding activities in brain membranes. Such antibodies will be used also in immunocytochemical analysis of kappa-opioid receptor distribution and compared with the reported distribution of putative kappa binding sites. The identity of the kappa-opioid receptor clone will be substantiated further by the isolation and sequencing of the 125I-B-endorphin-receptor complexes when labelling was carried out in the presence of mu- and delta- opioid ligands. The gene structure and the nucleotide sequence of the kappa-opioid receptor clone will be determined and compared with that of mu- and delta-opioid receptor clones. Deletion and nucleotide insertion mutation studies will be carried out to investigate the structural requirement for kappa-opioid receptor activities.