POSTTRANSCRIPTIONAL REGULATION OF SEROTONIN RECEPTORS

5-羟色胺受体的转录后调节

• 批准号: 2038657
• 负责人: Ronald B. Emeson
• 金额: $ 22.19万
• 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/2038657
• 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.

5-HT 2血清素受体家族的成员被认为是关键 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， 幻觉 此外，5-HT 2A和5-HT 2C受体都具有 与精神异常如精神抑郁症有关， 焦虑症和精神分裂症 最近的研究表明， 多种5-HT 2c受体亚型的产生受一种新的 RNA加工过程称为RNA编辑。 这篇文章- 转录修饰可以代表另外的机制， 这些细胞通过改变细胞外信号来调节它们的反应， 受体的功效：G蛋白相互作用;长期 拟议研究的目标是定义细胞 β-肾上腺素能信号调节机制 在中枢神经系统中的传导。 我们建议检查5-HT 2c受体亚型的功能， 通过在转染的NIH-3 T3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表征将 包括配体结合亲和力、组成型受体活化 脱敏动力学、肌醇磷酸水解和直接 受体检查：G蛋白相互作用和偶联 的特异性 将进行定点突变以定位 负责观察到的受体变化的关键残基 功能 负责顺式活性调节序列的鉴定 用于指示5-HT 2c受体RNA的位点特异性模式 处理将利用组织培养模型系统， 表现出与体内观察到的相似的RNA加工模式。 来自转染了一种新的细胞因子的大鼠C6胶质瘤细胞系的RNA分析 各种突变的5-HT 2cR转录单位，将作为主要的 这些绘图研究的方法。 体外RNA的开发 还将利用来自大鼠脑的核提取物进行编辑反应。 作为一种映射技术，通过测试体外 转录的RNA转录物被精确地修饰。 该体外 分析系统也将作为一种直接的生化方法， 参与的细胞机器的表征和纯化 这种转录后加工反应。 为了确定来自5-Ht 2b受体的RNA转录物是否 经历类似于5-HT 2cR所观察到的编辑事件， 将在基因组和cDNA之间进行核苷酸序列比较 克隆 单个cDNA分离物的序列分析和引物- 扩展策略，类似于为5-HT 2c研究开发的策略 转录本，将用于评估5-HT 2A和5-HT 2b RNA 处理. 编码5-HT 2A和5-HT 2b受体的RNA转录本 经历这样的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。 是 预计这些研究将提供新的见解， 调节参与转导的细胞过程， 多巴胺能信号和多种5-羟色胺受体的作用 神经系统