POSTTRANSCRIPTIONAL REGULATION OF SEROTONIN RECEPTORS

5-HT2 5-羟色胺受体家族的成员被认为起着关键作用 在许多生理和行为过程中的作用，包括 神经元兴奋性、摄食行为、昼夜节律以及 幻觉。此外，5-HT2A和5-HT2C受体都有 与精神病抑郁等精神异常有牵连， 焦虑和精神分裂症。最近的研究表明， 多种5-HT2C受体亚型的产生受一种新的 RNA处理事件称为RNA编辑。这篇帖子- 转录修饰可能代表一种额外的机制，通过 哪些细胞通过改变细胞外信号来调节它们对细胞外信号的反应 受体的功效：G蛋白相互作用；LING术语 拟议研究的目标是定义细胞 5-羟色胺能信号的调节机制 中枢神经系统中的转导。 我们建议研究产生的5-HT2C受体亚型的功能 在NIH-3T3成纤维细胞模型系统中进行RNA编辑。 多种受体异构体Will的药理学特征 包括配体结合亲和力、结构性受体激活 脱敏动力学、肌醇磷脂水解和直接 受体的检测：G蛋白相互作用和偶联 专一性。将进行定点突变以本地化 受体观察变化的关键残基(S) 功能。 识别顺式活性调控序列 用于决定5-HT2C受体RNA的位点特异性模式 处理将利用组织培养模型系统，该系统 显示出与体内观察到的类似的RNA处理模式。 转A基因大鼠C6胶质瘤细胞系的RNA分析 各种突变的5-HT2cR转录单位，将作为主要的 这些测绘研究的方法。体外核糖核酸的研制 利用大鼠脑核提取液的编辑反应也将被 作为一种作图技术，通过测试体外试验的能力 转录的RNA转录本需要准确修改。这是一种体外实验 化验系统也将作为一种直接的生化方法 细胞机械参与的特性和纯化 这种转录后加工反应。 为了确定RNA转录物是否来自5-HT2B受体 经历与为5-HT2cR观察到的事件类似的编辑事件， 基因组和cdna之间的核苷酸序列比较 克隆人。各分离株的序列分析及引物分析-- 扩展策略，类似于为研究5-HT2C而开发的策略 转录本，将用于评估5-HT2A和5-HT2B RNA 正在处理。编码5-HT2A和5-HT2B受体的RNA转录产物 经历了这样的RNA编辑事件，这些研究将扩展到 研究这种修饰对受体功能的影响。它是 预计这些研究将提供关于以下方面的新见解 参与转导的细胞过程的调节 5-羟色胺能信号及多种5-羟色胺受体在脑缺血中的作用(S) 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.