MOLECULAR BIOLOGY OF OPIATE RECEPTORS

阿片类药物被广泛用作镇痛剂，但其主要副作用之一- 成瘾的影响。 阿片类药物通过以下方式诱导其生物效应： 与三种主要亚型或受体相互作用，即μ、δ和 卡帕 脑啡肽和强啡肽相互作用 特别是δ和κ受体。 我们有 最近克隆了小鼠κ和δ阿片受体cDNA。 我们 建议使用这些cDNA来阐明作用的分子机制 内源性神经递质和临床上使用的 作用于这些受体以诱导它们的 生物效应。 两种阿片受体的氨基酸序列 60%相同，主要差异在N-末端区域。 我们 将使用定点突变来测试基于 诱变以鉴定κ和δ受体的结构域 与G蛋白相连，G蛋白将这些受体与腺苷酸环化酶偶联， Ca++和K+通道。 为了确定哪些G蛋白与 克隆阿片受体，我们将采用免疫沉淀技术 使用针对Gi和Go亚型的选择性抗血清来检测 特异性阿片受体/G蛋白复合物的细胞系，其中我们 稳定表达δ和κ受体。 我们还将使用 这些细胞系，以确定是否每个克隆阿片受体夫妇 Ca++和K+通道，采用膜片钳电生理 技术. 这些研究将确定阿片类药物的功能域 受体和与每个受体连接的细胞效应系统。 用δ或κ选择性激动剂长期治疗动物 诱导耐受性。 耐受性的分子基础与 阿片受体脱敏 研究δ和κ激动剂 在细胞水平诱导耐受，我们将确定生物化学 参与δ和κ受体脱敏的事件。 初始 研究表明，克隆的κ和δ受体 在激动剂治疗后脱敏， 肾上腺素能受体激酶（BARK）参与脱敏。 我们将确定这种激酶是否催化 阿片受体引起脱敏，并将研究 BARK是否也参与了δ和κ受体的下调， 调控 为了测试对慢性鸦片的神经适应性反应 阿片受体基因表达的变化，我们将确定 无论是对培养物中的细胞进行长期处理，还是对具有亚型 选择性阿片激动剂改变δ或κ受体mRNA水平 采用北方分析和原位杂交组织化学。 这些 研究将确定κ和δ受体的分子基础 脱敏和耐受性，并将研究长期的细胞 长期使用鸦片的后果。 本提案的总体目标是 是为了确定κ和δ阿片类物质 诱导生物学和临床效应，并将揭示 长期使用激动剂的副作用的分子基础 受体。

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.