POSTTRANSCRIPTIONAL REGULATION OF SEROTONIN RECEPTORS

5-HT2 血清素受体家族的成员被认为发挥着关键作用 在许多生理和行为过程中的作用，包括 神经元兴奋性、进食行为、昼夜节律等 幻觉。 此外，5-HT2A 和 5-HT2C 受体均具有 与精神异常有关，例如精神病性抑郁症， 焦虑症和精神分裂症。 最近的研究表明， 多种 5-HT2c 受体亚型的产生受一种新的调控 RNA加工事件称为RNA编辑。 这篇文章—— 转录修饰可能代表另一种机制 哪些细胞通过改变细胞外信号来调节其反应 受体的功效：G-蛋白相互作用；灵术语 拟议研究的目标是定义细胞 参与血清素信号调节的机制 中枢周围神经系统的转导。 我们建议检查所产生的 5-HT2c 受体亚型的功能 通过在转染的 NIH-3T3 成纤维细胞模型系统中进行 RNA 编辑。 多种受体亚型的药理学特征将 包括配体结合亲和力、组成型受体激活、 脱敏动力学、磷酸肌醇水解和直接 受体检查：G-蛋白相互作用和偶联 特异性。 将进行定点诱变以定位 导致观察到的受体变化的关键残基 功能。 鉴定负责的顺式活性调节序列 用于指示 5-HT2c 受体 RNA 的位点特异性模式 加工将利用组织培养模型系统， 表现出与体内观察到的类似的 RNA 加工模式。 转染大鼠 C6 神经胶质瘤细胞系的 RNA 分析 多种突变 5-HT2cR 转录单位，将作为主要 这些绘图研究的方法。 体外 RNA 的开发 利用大鼠大脑的核提取物进行编辑反应也将是 通过测试体外能力用作绘图技术 转录的RNA转录物被精确修饰。 这在体外 测定系统也将作为一种直接的生化方法，允许 参与的细胞机器的表征和纯化 此类转录后加工反应。 确定 RNA 转录物是否源自 5-Ht2b 受体 经历类似于 5-HT2cR 观察到的编辑事件， 将在基因组和 cDNA 之间进行核苷酸序列比较 克隆。 单个 cDNA 分离物和引物的序列分析 延伸策略，类似于 5-HT2c 研究开发的策略 转录本，将用于评估 5-HT2A 和 5-HT2b RNA 处理。 编码 5-HT2A 和 5-HT2b 受体的 RNA 转录物 经历此类RNA编辑事件后，这些研究将扩展到 检查此类修饰对受体功能的影响。 它是 预计这些研究将提供有关 参与转导的细胞过程的调节 血清素信号和多种血清素受体的作用 神经系统。

Members of the 5-HT2 serotonin receptor family are thought to play key roles in a number of physiological and behavioral processes, including neuronal excitability, feeding behavior, circadian rhythms, and hallucinations. In addition, both the 5-HT2A and 5-HT2C receptors have been implicated in mental abnormalities such as psychotic depression, anxiety and schizophrenia. Recent studies have indicated that the generation of multiple 5-HT2c receptor isoforms is regulated by a novel RNA processing event referred to as RNA editing. This post- transcriptional modification may represent an additional mechanism by which cells modulate their response to extracellular signals by altering the efficacy of receptor: G-protein interactions; the ling term objectives of the proposed research are to define the cellular mechanisms involved in the regulation of serotonergic signal transduction in the central peripheral nervous systems. We propose to examine the function of 5-HT2c receptor isoforms generated by RNA editing in a transfected NIH-3T3 fibroblast model system. Pharmacological characterization of multiple receptor isoforms will include ligand binding affinities, constitutive receptor activation, desensitization kinetics, phosphoinositide hydrolysis and direct examinations of receptor: G-protein interactions and coupling specificity. Site-directed mutagenesis will be performed to localize the key residue(s) responsible for observed changes in receptor function. Identification of the the cis-active regulatory sequences responsible for dictating the site-specific patterns of 5-HT2c receptor RNA processing will take advantage of tissue culture model systems which exhibit RNA processing patterns analogous to those observed in vivo. Analyses of RNA from the rat C6 glioma cell line, transfected with a variety of mutant 5-HT2cR transcription units, will serve as the primary methodology for these mapping studies. Development of an in vitro RNA editing reaction utilizing nuclear extracts from rat brain will also be used as a mapping technique by testing the ability of in vitro transcribed RNA transcripts to be accurately modified. This in vitro assay system will also serve as a direct bio-chemical approach allowing characterization and purification of the cellular machinery involved in such post-transcriptional processing reactions. To determine if RNA transcripts derived from the 5-Ht2b receptors undergo editing events similar to those observed for the 5-HT2cR, nucleotide sequence comparisons will be made between genomic and cDNA clones. Sequence analysis of individual cDNA isolates and primer- extension strategies, similar to those developed for studies of 5-HT2c transcripts, will be employed to assess 5-HT2A and 5-HT2b RNA processing. Should RNA transcripts encoding 5-HT2A and 5-HT2b receptors undergo such RNA editing events, these studies will be extended to examine the effect to such modifications on receptor function. It is anticipated that these studies will provide new insights concerning the regulation of cellular processes involved in the transduction of serotonergic signals and the role(s) of multiple serotonin receptors in the nervous system.