PROTEIN(S) INVOLVED IN NEUROTRANSMISSION

参与神经传递的蛋白质

• 批准号: 3375999
• 负责人: Jean Chen Shih
• 金额: $ 25.64万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-03-01 至 1996-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3375999
• 关键词: DNA footprinting; RNase protection assay; animal tissue; brain; chloramphenicol acetyltransferase; genetic promoter element; genetic regulation; human tissue; in situ hybridization; laboratory rat; molecular cloning; neural transmission; neurotransmitters; postmortem; protein kinase C; protein structure function; reporter genes; restriction mapping; second messengers; serotonin receptor; transcription factor

5-hydroxytryptamine (5-HT, serotonin) and its receptors have been implicated in the etiology and therapeutic treatment of human mental depression. A basic knowledge of 5-HT receptors will help to better understand the molecular mechanism of 5-HT mediated neural function and will provide insights into the molecular basis of mental depression. The objectives of this project are to investigate the structure and function relationships, the genomic organization and the regulation of human 5-HT-2 receptor(s). These objectives will be accomplished by using the rat brain 5-HT-2 receptor cDNA cloned recently. The specific aims of the proposed study are outlined below. 1. To clone 5-HT-2 receptor and related cDNAs from human brain. Various 5-HT-2 receptor subtypes and related cDNA clones will be isolated and sequenced. The pharmacological profiles and the second messenger systems linked to the expressed receptors (phosphoinositol (PI) turnover, CA++ release, or adenylate cyclase) will be characterized. 2. To map the chromosomal location of the 5-HT-2 receptor gene. This aim will be accomplished by somatic cell mapping and in situ hybridization of the chromosome. 3. To isolate human genomic 5-HT-2 receptor clones. 4. To construct the restriction map and to sequence human 5-HT-2 receptor genomic clones. 5. To identify and to analyze promoter regions of this gene. This aim will be accomplished by RNase protection, primer extension, and chloramphenicol acetyltransferase (CAT) reporter gene assay. The cis- elements, trans-acting factors and the functions of these elements will be systematically analyzed. 6a. To investigate if 5-HT-2 receptor gene is regulated by protein kinase C (PKC) and/or protein kinase A (PKA). The effects of TPA (12-0- tetradecanoyl-phorbol-14 acetate, activator of PKC) on the 5-HT-2 receptor gene expression will be studied by using CAT report gene assay and footprinting analysis. The ability of 1-(5-isoquinolylsulfonyl)-2- methylpiperazine (H7) to block TPA activation will be studied. Similar studies will be performed using activators of PKA such as forskolin and 8- bromodibutyryl cAMP. 6b. To investigate if antagonist induced 5-HT-2 receptor down regulation is at the transcriptional level.

5-羟色胺（5-HT，血清素）及其受体已被 与人类精神疾病的病因学和治疗有关 沮丧。 5-HT 受体的基本知识将有助于更好地 了解5-HT介导的神经功能的分子机制和 将提供对精神抑郁症分子基础的见解。 这 该项目的目标是研究结构和功能 关系、基因组组织和人类 5-HT-2 的调节 受体。 这些目标将通过使用大鼠大脑来实现 最近克隆了5-HT-2受体cDNA。 拟议的具体目标 研究概述如下。 1. 克隆人脑5-HT-2受体及相关cDNA。 各种各样的 5-HT-2受体亚型和相关cDNA克隆将被分离并 已测序。 药理学特征和第二信使系统 与表达的受体相关（磷酸肌醇 (PI) 周转率，CA++ 释放或腺苷酸环化酶）将被表征。 2. 绘制5-HT-2受体基因的染色体位置图。 这个目标 将通过体细胞作图和原位杂交来完成 染色体。 3.分离人类基因组5-HT-2受体克隆。 4. 构建限制性图谱并对人5-HT-2受体进行测序 基因组克隆。 5. 鉴定并分析该基因的启动子区域。 这个目标 将通过 RNase 保护、引物延伸和 氯霉素乙酰转移酶（CAT）报告基因测定。 顺式 元素、反式作用因子以及这些元素的功能将被 进行了系统分析。 6a. 研究5-HT-2受体基因是否受蛋白激酶调控 C (PKC) 和/或蛋白激酶 A (PKA)。 TPA 的影响 (12-0- 5-HT-2 受体上的十四烷酰佛波醇 14 乙酸酯，PKC 激活剂 将使用 CAT 报告基因分析来研究基因表达 足迹分析。 1-(5-异喹啉磺酰基)-2-的能力 将研究甲基哌嗪 (H7) 阻断 TPA 活化的作用。 相似的 将使用 PKA 激活剂（例如毛喉素和 8-）进行研究 溴二丁酰 cAMP。 6b. 研究拮抗剂是否诱导 5-HT-2 受体下调 是在转录水平。