HORMONAL REGULATION OF B-ADRENERGIC RECEPTORS

B-肾上腺素能受体的激素调节

• 批准号: 2224222
• 负责人: SULEIMAN WAKIM BAHOUTH
• 金额: $ 10.28万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-02-18 至 1997-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2224222
• 关键词: DNA footprinting; G protein; beta adrenergic receptor; chloramphenicol acetyltransferase; enzyme activity; gel mobility shift assay; gene expression; genetic promoter element; genetic transcription; heart cell; hormone regulation /control mechanism; laboratory rat; newborn animals; nucleic acid sequence; receptor coupling; retinoids; thyroid hormones; transfection

G protein-coupled receptors comprise a large family of receptors which interact with diverse chemical structures to activate GTP-binding regulatory proteins. The objective of the proposed studies is to determine the mechanism by which the expression of these receptors is regulated, using the beta-adrenergic receptor (beta-AR) as the model. The beta-AR appears to be a dominant receptor for mediating the functions of catecholamines in the heart, which include regulation of heart rate, force and rhythmic contractions. The overall density of cardiac beta-AR in vivo and in cultured ventricular myocytes (VM) in vitro is increased by thyroid hormone (T3). T3 increased the number, mRNA level, and gene transcription of beta1-AR in VM. The transcriptional effects of T3 are mediated by nuclear T3 receptors (T3R) which bind to T3 response elements (TRE) in T3 responsive genes. The major goal of this proposal is to characterize beta- AR expression in VM. The primary hypothesis underlying this proposal is that T3 increases cardiac beta-AR expression by a transcriptional mechanism orchestrated by heterodimer formation between nuclear T3R and auxiliary nuclear proteins at the TRE in the promoter region of the beta1-AR gene. The aim of this proposal is to identify the TREs in the beta1-AR gene and to characterize the proteins that bind to the TRE and stimulate the transcription of the beta1-AR gene. Neonatal rat VM will be used to study the regulation of cardiac beta-AR by T-3. The first aim is to identify the DNA sequences (TREs) in the beta1-AR gene that confer transcriptional regulation by T3. Genomic fragments from the 5' flanking region of the beta1-AR gene will be ligated to chloramphenicol acetyl transferase (CAT) gene to generate chimeric beta1-AR CAT vectors. These vectors will be transiently transfected into VM, followed by measuring the effect of T3 on CAT activity. In the second aim, we will examine DNA-protein interactions in the promoter regulatory region of the beta1-AR gene. The interaction between DNA sequences in the beta1-AR promoter and rat heart nuclear proteins will be characterized by means of DNAse I footprint analysis to define those domains that bind nuclear proteins. Gel mobility shift assays with purified T3R and nuclear proteins will be used to evaluate their specificity in binding to the beta1-AR TRE. In the third aim, we will identify the proteins that bind to the beta1-AR TRE and heterodimerize with the T3R to enhance hormonal transactivation of beta1-AR gene. These experiments will involve the use of gel mobility assays an other related techniques to characterize the T3R binding partner at the beta1-AR TRE. The functional significance of these proteins will be determined by transient transfection assays to confirm their role in the transcriptional regulation by T3. Other experiments will determine the role of retinoic acid and its nuclear receptor in regulating beta1-AR gene expression. Characterization of the beta1-AR TRE and the proteins that bind to it, is an important step in elucidating the mechanism by which T3 directly stimulates transcription. These studies will be relevant to regulation of many G protein-linked receptor genes whose expression is controlled transcriptionally by hormones.

G蛋白偶联受体包括一个大型受体家族 与各种化学结构相互作用以激活GTP结合 调节蛋白。 拟议研究的目的是确定 调节这些受体表达的机制 使用β-肾上腺素能受体（β-AR）作为模型。 beta-ar 似乎是介导功能的主要受体 心脏中的儿茶酚胺包括心率调节，力 和有节奏的收缩。 体内心脏β-AR的总密度 在体外培养的心室肌细胞（VM）中，甲状腺增加了 激素（T3）。 T3增加了数量，mRNA水平和基因转录 VM中的Beta1-ar。 T3的转录效应是由 核T3受体（T3R）与T3中的T3反应元件（TRE）结合 响应基因。 该提议的主要目标是表征β- VM中的AR表达。 该提议的主要假设是 T3通过转录机制增加心脏β-AR表达 由核T3R和辅助辅助二聚体编排的策划 beta1-ar基因启动子区域的TRE的核蛋白。 该提议的目的是确定beta1-ar基因中的TRE和 表征与TRE结合的蛋白质并刺激 beta1-ar基因的转录。 新生儿大鼠VM将用于研究 T-3对心脏β-AR的调节。 第一个目的是确定 beta1-ar基因中的DNA序列（TRE）赋予转录 T3调节。 来自5'侧面区域的基因组片段 Beta1-AR基因将连接到氯霉素乙酰基转移酶（CAT） 基因生成嵌合beta1-ar猫矢量。 这些向量将是 瞬时转染到VM中，然后测量T3对 猫活动。 在第二个目标中，我们将检查DNA-​​蛋白质相互作用 在beta1-ar基因的启动子调节区域中。 相互作用 在beta1-ar启动子和大鼠心脏核中的DNA序列之间 蛋白质将通过DNase I足迹分析的方式来表征 定义那些结合核蛋白的结构域。 凝胶移动转移测定法 纯化的T3R和核蛋白将用于评估其 与beta1-ar tre结合的特异性。 在第三个目标中，我们将 识别与beta1-ar tre结合的蛋白质并与 T3R增强β1-AR基因的激素反式激活。 这些 实验将涉及凝胶迁移率分析的使用 在Beta1-AR Tre中表征T3R结合伙伴的技术。 这些蛋白质的功能意义将由 瞬态转染测定法以确认其在转录中的作用 T3调节。 其他实验将决定视网膜的作用 酸及其在调节β1-AR基因表达的核受体。 beta1-ar tre的表征和与之结合的蛋白质是 阐明直接T3的机制的重要步骤 刺激转录。 这些研究将与调节 许多G蛋白连接受体基因的表达受到控制 通过激素转录。