CLONING AND CHARACTERIZATION OF CANNABINOID RECEPTORS

大麻是目前被滥用最广泛的街头毒品。然而， 大麻素受体系统在健康和健康中的功能意义 疾病包括使用大麻素作为镇痛剂，止吐剂 癌症患者，抗癫痫药和抗青光眼药，如 以及免疫调节剂。这些不同的药理作用 大麻素暗示了受体亚型的可能性。二 到目前为止，已经确定了大麻素受体；其中一个位于 主要在中枢神经系统(CB1)，而另一种是 在外周组织中表达(CB2)。这个项目的目标是 大麻素受体亚型的药理学特征 以阐明受体在体内的作用。我们将稳定使用 表达人的cDNA克隆的转基因细胞系 受体亚型提供纯净、同质的受体群体 用于筛选候选药物。在具体目标1中，约束和信号 表达CB1和CB2基因的转基因细胞的转导特性 将对大麻素受体亚型进行表征。这些研究将 识别区分受体亚型的选择性配体 结合和功能分析中的受体亚型。分子 激动剂与大麻素受体结合的机制不是 为人所知。特定目标2将检查CB1或CB2的哪些域 受体识别配体并激活第二信使 系统。这些研究将通过定点突变和 构建CB1/CB2嵌合受体以定位区域 对受体识别至关重要的受体cDNA和第二 信使功能。在具体目标3中，额外推定的大麻素 用聚合酶链式反应和文库技术分离受体 筛选技术。这些研究将使用累积的信息 从目前分离的受体克隆中获得以分离其他 子类型，这些子类型可以为隔离其他 大麻素受体亚型。总之，这些实验描述了一种 研究大麻素受体功能的分子生物学方法。 这些研究将有助于设计新的治疗策略。 涉及大麻素系统。此外，对分子的洞察 激活大麻素受体的机制可能会导致更好的 对人类滥用大麻素的理解。

Marijuana is currently the most widely abused street drug. However, the functional significance of the cannabinoid receptor system in health and disease includes the use of cannabinoids as analgesics, antiemetics in cancer patients, anticonvulsant for epilepsy and as antiglaucoma agents as well as immunomodulatory agents. These diverse pharmacological effects of cannabinoids suggest the possibility of receptor subtypes. Two cannabinoid receptors have been identified to date; one is located predominantly in the central nervous system (CB1), whereas the other is expressed in peripheral tissues (CB2). The goal of this project is to characterize the pharmacology of subtypes of the cannabinoid receptor in order to elucidate the role of the receptors in vivo. We will use stably transfected cell lines which express cDNA clones of the individual receptor subtypes to provide a pure, homogeneous population of receptors for screening drug candidates. In specific aim 1, the binding and signal transduction properties of transfected cells expressing CB1 or CB2 cannabinoid receptor subtypes will be characterized. These studies will identify receptor subtype-selective ligands which discriminate between the receptor subtypes in binding and functional assays. The molecular mechanisms by which agonists bind to the cannabinoid receptors are not known. Specific aim 2 will examine which domains of the CB1 or CB2 receptors recognize the ligands and which activate second messenger systems. These studies will be conducted by site-directed mutagenesis and the construction of CB1/CB2 chimeric receptors in order to map the regions of the receptor cDNAs critical to receptor recognition and second messenger function. In specific aim 3, additional putative cannabinoid receptors will be isolated by polymerase chain reaction and library screening techniques. These studies will use the cumulative information obtained from the currently isolated receptor clones to isolate other subtypes which may provide data for the isolation of yet additional cannabinoid receptor subtypes. In summary, these experiments describe a molecular biological approach to studying cannabinoid receptor function. These studies will facilitate the design of new therapeutic strategies involving the cannabinoid system. Furthermore, insight into the molecular mechanisms of activation of cannabinoid receptors may lead to a better understanding of cannabinoid abuse in humans.