POSTTRANSCRIPTIONAL REGULATION OF SEROTONIN RECEPTORS

5-羟色胺受体的转录后调节

• 批准号: 2655549
• 负责人: Ronald B. Emeson
• 金额: $ 22.72万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2655549
• 关键词: 3T3 cells; G protein; animal genetic material tag; biological signal transduction; complementary DNA; genetic regulatory element; inositol phosphates; laboratory rat; nucleic acid sequence; posttranscriptional RNA processing; protein isoforms; receptor coupling; receptor expression; serotonin receptor

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.

人们认为5-HT2 5-羟色胺受体家族的成员会玩钥匙 在许多生理和行为过程中的角色，包括 神经元兴奋性，喂养行为，昼夜节律和 幻觉。 此外，5-HT2A和5-HT2C受体都具有 与精神异常有关，例如精神病性抑郁症， 焦虑和精神分裂症。 最近的研究表明 多个5-HT2C受体同工型的产生受新颖的调节 RNA处理事件称为RNA编辑。 这个帖子 - 转录修改可能代表一个附加机制 哪些细胞通过改变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 涉及血清素能信号调节的机制 中央周围神经系统的转导。 我们建议检查产生的5-HT2C受体同工型的功能 通过转染的NIH-3T3成纤维细胞模型系统中的RNA编辑。 多种受体同工型的药理表征将 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 受体的检查：G蛋白相互作用和耦合 特异性。 将进行定向诱变以本地化 负责观察到的受体变化的关键残留物 功能。 识别负责的顺式调节序列 用于指示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 表现出类似于体内观察到的RNA处理模式。 从大鼠C6胶质瘤细胞系中RNA的分析，用A转染 多种突变体5-HT2CR转录单元将作为主要 这些映射研究的方法。 体外RNA的发展 利用大鼠大脑核提取物的编辑反应也将是 通过测试体外的能力，用作映射技术 转录的RNA转录本被准确修改。 这在体外 测定系统还将作为一种直接的生物化学方法 涉及的细胞机械的表征和纯化 这种转录后处理反应。 确定RNA转录本是否源自5-HT2B受体 经历与5-HT2CR观察到的事件类似的编辑事件， 核苷酸序列比较将在基因组和cDNA之间进行 克隆。 序列分析单个cDNA分离株和引物 扩展策略，类似于用于研究5-HT2C的策略 成绩单将用于评估5-HT2A和5-HT2B RNA 加工。 RNA转录本应该编码5-HT2A和5-HT2B受体 进行此类RNA编辑事件，这些研究将扩展到 检查这种修饰对受体功能的影响。 这是 预计这些研究将提供有关该研究的新见解 调节涉及转导的细胞过程 血清素能信号和多种5-羟色胺受体的作用 神经系统。