PROMOTER FUNCTION OF BETA 1 ADRENERGIC RECEPTOR GENES

Beta 1 肾上腺素能受体基因的启动子功能

• 批准号: 2191824
• 负责人: THOMAS FRIELLE
• 金额: $ 10.36万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2191824
• 关键词: RNA splicing; RNase protection assay; beta adrenergic receptor; clone cells; gene deletion mutation; genetic promoter element; genetic transcription; heart cell; human tissue; luciferin monooxygenase; molecular cloning; polymerase chain reaction; receptor expression; reporter genes; site directed mutagenesis; transfection

In the normal human heart, cardiac rate and contractility are stimulated primarily through signal transduction pathways which involve the beta-1- adrenergic receptor (beta-1-AR). The sympathetic neurotransmitter norepinephrine binds to the beta-1-AR which by activating the GTP-binding protein, GS, modulates cardiac Na+, Ca++ channels and the sinoatrial pacemaker current by both direct and cAMP-dependent mechanisms. In the failing human heart, a reduction in the density of the beta-1-AR, in part, severely limits cardiac function, possibly as a protective mechanism. The mechanisms which modulate beta-1-adrenergic receptor expression are clearly of great physiological significance considering the central role the beta-1-AR occupies in the functioning of the normal and the failing heart. We have recently cloned and sequenced the entire coding region of the human beta-1-adrenergic receptor gene and over 3 kb of the 5' promoter region. Whereas amino acid sequences of the human beta-1-AR and the beta-2-AR are 75% identical, no sequence homology is found when the two promoter regions are compared. This suggests that the transcription of each beta-AR subtype is regulated by different mechanisms. The beta-1-AR promoter contains consensus sites for insulin (ISRE), glucocorticoid (GRE), estrogen/thyroid (ERE/TRE), cAMP (CRE) and nerve growth factor (NGFI) dependent regulation. Also present are consensus sites for the cardiac myocyte specific E-box, CArG box and M-CAT transcriptional elements and a binding site for MEF-2, a cardiac specific transcription factor all of which suggest a cardiac myocyte-specific transcriptional regulation of the beta-1-AR. Transcriptional regulation conferred by these elements and by as yet unidentified regulatory sequences will be examined by assessing the ability of mutated and deleted (5',3' and internal) beta-1-AR promoter regions to drive transcription of the luciferase reporter gene. In particular, NGF-dependent and cardiac myocyte-specific transcription will be studied using appropriately mutated promoter sequences to transect neuronal cardiac myocyte and control cells. Using nested deletions at the 5' end of the promoter, we have demonstrated the existence of both positive and negative regulatory regions which modulate luciferase expression in rat C6 glioma cells. Internal deletions within these 5' regions have been constructed to more precisely localized transcriptionally important elements and will be assayed for transcriptional activity in all appropriate cell lines. A consensus CCAAT box at -366 bp and CCAAT and TATA boxes at -1400 bp suggest the existence of two transcriptional start sites. Northern analysis of heart mRNA, RNAse protection and primer extension analyses all suggest the existence of two alternate promoters, one of which may require splicing within the 5' noncoding region. PCR amplification of cDNAs synthesized from the 5' termini of poly(A+ ) mRNAs will be used to conclusively identify alternate promoters. The tissue specific use of these alternate promoters will also be determined using a combination of Northern and RNAse protection analyses of poly(A+ ) mRNA isolated from various human tissues and promoter dependent luciferase expression in appropriate cell lines.

在正常的人心脏中，刺激心脏率和收缩力 主要通过涉及β-1-的信号转导途径 肾上腺素能受体（β-1-AR）。 交感神经递质 去甲肾上腺素与β-1-AR结合，该AR通过激活GTP结合 蛋白质，GS，调节心脏NA+，Ca ++通道和辛里尔 起搏器通过直接和cAMP依赖机制的电流。 在 失败的人心，降低了β-1-AR密度的降低 部分，严重限制心脏功能，可能是保护性 机制。 调节β-1-肾上腺素受体的机制 考虑到表达显然具有很大的生理意义 Beta-1-AR在正常功能中占据的核心作用 和失败的心。 我们最近克隆并测序了整个编码区域 人β-1-肾上腺素能受体基因和超过3 kb的5'启动子 地区。 而人β-1-AR的氨基酸序列和 beta-2-ar是75％相同的，当两者都没有发现序列同源 比较启动子区域。 这表明转录 每个β-AR亚型都受不同机制调节。 beta-1-ar 启动子包含胰岛素（ISRE）的共识位点，糖皮质激素 （GRE），雌激素/甲状腺（ERE/TRE），CAMP（CRE）和神经生长因子 （NGFI）依赖调节。 同样存在的是 心肌细胞特异性电子盒，carg盒和M-CAT转录 MEF-2的元素和结合位点，心脏特异性转录 所有这些都表明心肌细胞特异性转录 Beta-1-AR的调节。 这些元素赋予的转录调节 未识别的监管序列将通过评估 突变和删除（5'，3'和内部）beta-1-ar启动子的能力 驱动荧光素酶报告基因转录的区域。 在 特别是，依赖NGF和心肌细胞特异性转录将 使用适当突变的启动子序列进行横切研究 神经元心肌细胞和对照细胞。 使用嵌套删除 发起人的5'末端，我们已经证明了两者的存在 调节荧光素酶的正和负调节区域 大鼠C6胶质瘤细胞中的表达。 这5'内的内部删除 区域已被构建为更精确的本地化 转录重要的元素，并将分析 在所有适当的细胞系中的转录活性。 一个共识CCAAT盒子，位于-366 bp，CCAAT和TATA盒子-1400 bp 建议存在两个转录起始位点。 北方 心脏mRNA，RNase保护和底漆扩展分析的分析 所有人都表明存在两个替代启动子，其中一个可能 需要在5'非编码区域内进行剪接。 PCR扩增 从poly（a+）mRNA的5'末端合成的cDNA将用于 最终确定替代启动子。 组织特定的用途 这些替代启动子也将使用 从从中分离出的poly（a+）mRNA的北部和RNase保护分析 各种人体组织和启动子依赖性荧光素酶的表达 适当的单元线。