MOLECULAR BIOLOGY OF OPIATE RECEPTORS

鸦片类药物被广泛用作止痛剂，但它们的主要副作用之一-- 对上瘾的影响。阿片类药物通过以下途径产生生物学效应 与三个主要亚型或受体相互作用，即Mu、Delta和 卡帕。神经肽、脑啡肽和强啡肽相互作用 分别与Delta和kappa受体结合。我们有 最近克隆了小鼠kappa和Delta阿片受体基因。我们 建议利用这些cdna来阐明其作用的分子机制。 内源性神经递质和临床使用的 作用于这些受体的药理制剂，以诱导其 生物效应。两种阿片受体的氨基酸序列 与N-末端区域的主要差异有60%的同源性。我们 将使用定点突变来检验基于 用突变技术鉴定Kappa和Delta受体的结构域 与G蛋白相连，G蛋白将这些受体与腺苷环化酶偶联， Ca++和K+通道。确定哪些G蛋白与 克隆阿片受体，我们将使用免疫沉淀技术 用选择性抗GI和GO亚型抗血清检测 来自细胞系的特异性阿片受体/G蛋白复合体 稳定表达德尔塔和kappa受体。我们还将使用 这些细胞系确定每个克隆的阿片受体是否成对 膜片钳电生理对钙、钾通道的作用 技巧。这些研究将确定阿片剂的功能区域。 受体和连接到每个受体的细胞效应系统。 用Delta或Kappa选择性激动剂慢性治疗动物 能产生宽容。耐受性的分子基础已经被认为是 阿片受体脱敏。研究Delta和Kappa激动剂 在细胞水平上诱导耐受，我们将鉴定生化 参与Delta和kappa受体脱敏的事件。在首字母中 研究表明，克隆的kappa和Delta受体 在激动剂治疗后脱敏，酶-β- 肾上腺素能受体激酶(Bark)参与了脱敏反应。 我们将确定该激酶是否催化磷酸化 阿片受体导致脱敏并将进行调查 树皮是否也参与了三角洲和kappa受体的下调- 监管。测试对慢性阿片类药物的神经适应性反应 利用阿片受体基因表达的变化，我们将确定 无论是长期处理培养中的细胞还是患有亚型的啮齿动物 选择性阿片受体激动剂改变β或kappa受体mRNA水平 采用Northern分析和原位杂交组织化学方法。这些 研究将确定kappa和Delta受体的分子基础 脱敏和耐受性，并将研究长期细胞 长期使用鸦片的后果。这项提案的总体目标是 是确定kappa和Delta鸦片的细胞机制 诱导三种生物学和临床效应，并将揭示 中枢神经系统长期使用兴奋剂副作用的分子基础 感受器。

Opiates are widely used as analgesic agents, but one of their major side- effects in addiction. Opiates induce their biological effects by interacting with three major subtypes or receptor, the mu, delta, and kappa. The neuropeptide, enkephalins and dynorphins interact specifically with delta and kappa receptors, respectively. We have recently cloned the mouse kappa and delta opiate receptor cDNA. We propose to use these cDNA to elucidate the molecular mechanisms of action of the endogenous neurotransmitters and of the clinically used pharmacological agents that act on these receptors to induce their biological effects. The amino acid sequences of the two opiate receptors are 60% identical with major differences in the N-terminal regions. We will use site-directed mutagenesis to test the hypothesis based mutagenesis to identify the domains of the kappa and delta receptors linked to G proteins, which couple these receptors to adenylyl cyclase, Ca++ and K+ channels. To determine which G proteins associate with the cloned opiate receptors, we will employ an immunoprecipitation technique using selective antisera against the subtypes of Gi and Go to detect specific opiate receptor/G protein complexes from cell lines in which we have stably expressed the delta and kappa receptors. We will also use these cell lines to determine whether each cloned opiate receptor couples to Ca++ and K+ channels employing patch-clamp electrophysiological techniques. These studies will identify functional domains of the opiate receptors and the cellular effector systems linked to each receptor. Chronic treatment of animals with delta or kappa selective agonists induces tolerance. The molecular basis of tolerance has been associated with opiate receptor desensitization. To study delta and kappa agonist induced tolerance at a cellular level, we will identify the biochemical events involved in delta and kappa receptor desensitization. In initial studies, we have shown that the cloned kappa and delta receptors desensitize following agonist treatment and that the enzyme beta- adrenergic receptor kinase (BARK) is involved in the desensitization. We will determine whether this kinase catalyzes the phosphorylation of the opiate receptors to cause desensitization and will investigate whether BARK is also involved in delta and kappa receptor down- regulation. To test whether a neuroadaptive response to chronic opiate use is changes in opiate receptor gene expression, we will determine whether prolonged treatment of cells in culture or rodents with subtype selective opiate agonists alters delta or kappa receptor mRNA levels using Northern analysis and in situ hybridization histochemistry. These studies will determine the molecular basis of kappa and delta receptor desensitization and tolerance and will investigate the long-term cellular consequences of chronic opiate use. The overall goal of this proposal is to identify the cellular mechanisms by which kappa and delta opiate induce there biological and clinical effects and will reveal the molecular basis for the side-effects of chronic use of agonists at the receptors.