MOLECULAR BASIS OF TYROSINE HYDROXYLASE REGULATION

酪氨酸羟化酶调节的分子基础

• 批准号: 3405402
• 负责人: Dona M Chikaraishi
• 金额: $ 21.48万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3405402
• 关键词: RNA; RNA splicing; acetylcholinesterase; catecholamines; clone cells; complementary DNA; cyclic AMP; enzyme induction /repression; enzyme mechanism; epidermal growth factor; gene expression; genetic promoter element; genetic transcription; genetically modified animals; glucocorticoids; hormone receptor; laboratory mouse; laboratory rabbit; laboratory rat; messenger RNA; molecular cloning; neurochemistry; neurotransmitter metabolism; nicotinic receptors; nucleic acid sequence; organ culture; phosphorylation; posttranscriptional RNA processing; posttranslational modifications; sympathetic ganglion; tyrosine 3 monooxygenase

Tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine biosynthesis, is regulated by factors that increase its rate of synthesis (induction) and its intrinsic enzyme activity by post-transnational phosphorylation (activation). This proposal addresses both aspects of regulation. The mechanism by which various known inducers (cAMP, glucocorticoids, epidermal growth factor (EGF)) and environmental factors such as stress (transsynaptic induction) or trophic molecules induce TH RNA in vivo or in cultured cells will be studied. In particular, experiments will determine whether inductive factors responsible for the phenotypic switch in transmitter synthesis increase TH transcription rates or changes TH mRNA levels by post-transcriptional mechanism. Experiments will define cis-acting DNA sequences responsible for hormone or tissue-specific expression. These combine analyses of the functional DNA sties by transfection into cell lines and the ability of trans-acting factors to bind these sites. To correlate whether putative sites affect in vivo function, transient transfections will be performed in the presence of excess competing DNA encompassing the potential site, which would be expected to block hormone induction or tissue-specificity is blocked. Tissue specificity will also be assessed in vivo using transgenic mice bearing putative tissue-specific enhancers directing bacterial chloramphenicol acetyltransferase (CAT). The possibility that alternative splicing generates distinct TH molecules that can be differentially activated by phosphorylation will be explored by cloning cDNAs for various putative isoforms and determining their tissue distribution and inducibility by various manipulations. These isoforms will be individually expressed and their ability to be phosphorylated and activated by purified kinases in vitro will be assessed.

酪氨酸羟化酶（Th），速率限制酶中的酶 儿茶酚胺生物合成，受到增加的因素的调节 其合成速率（诱导）及其内在酶活性 通过转换后磷酸化（激活）。 这个建议 解决调节的两个方面。 各种已知诱导者的机制（营地， 糖皮质激素，表皮生长因子（EGF）和环境 诸如应力（透射性诱导）或营养等因素 分子在体内或培养细胞中诱导Th RNA 研究。 特别是，实验将确定是否 电感因素负责表型转换 发射机合成提高了TH转录率或变化 通过转录后机制，mRNA水平。 实验将定义负责的顺式作用DNA序列 激素或组织特异性表达。 这些结合了分析 功能性DNA通过转染到细胞系和 跨性因子结合这些位点的能力。 相关 推定位点是否影响体内功能，瞬态 转染将在竞争过多的情况下进行 DNA涵盖了潜在地点，这将有望 阻塞激素诱导或组织特异性被阻断。 组织 还将使用转基因小鼠在体内评估特异性 轴承假定的组织特异性增强剂指导细菌 氯霉素乙酰转移酶（CAT）。 替代剪接产生不同的可能性 可以通过磷酸化差异激活的分子 将通过克隆cDNA来探索各种推定的同工型，并且 确定其组织分布和各种诱导性 操纵。 这些同工型将单独表达，并且 它们通过纯化的磷酸化和激活的能力 将评估体外激酶。