MOLECULAR ENDOCRINOLOGY OF PEPCK GENE REGULATION BY CAMP

CAMP 对 PEPCK 基因调控的分子内分泌学

基本信息

批准号：
3245380
负责人：
PATRICK G QUINN
金额：
$ 17.93万
依托单位：
PENNSYLVANIA STATE UNIV HERSHEY MED CTR
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-05-01 至 1996-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3245380
关键词：
DNA binding protein RNA splicing complementary DNA cyclic AMP dimer fusion gene gene expression gene mutation genetic promoter element genetic regulatory element hormone regulation /control mechanism isomer phosphoenolpyruvate carboxylase site directed mutagenesis tissue /cell culture transcription factor transfection

项目摘要

The long-term objective of the research program described here is to understand how cAMP regulates gene expression with particular emphasis on the mechanism of action of the cAMP response element (CRE) binding protein (CREBP) and the interplay of factors regulating expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene. The CRE plays an essential role in both basal and cAMP-stimulated transcription of the PEPCK gene. Regulated expression of PEPCK, which catalyzes the rate-limiting step in gluconeogenesis, is essential for maintaining appropriate blood glucose concentrations. Gene transcription is regulated by the interaction of trans-acting protein factors with each other and RNA polymerase, which is facilitated by binding of these factors to cis-acting DNA elements. Cyclic AMP activates protein kinase A, resulting in phosphorylation of several proteins, including the CRE binding protein. CREBP has recently been purified, cloned and shown to be directly involved in regulation of gene expression. In order to understand how CREBP interacts with other factors to regulate gene expression, it is necessary to identify the domains of CREBP that are involved in transactivation. CREBP binds to the CRE as a dimer, and two isoforms that differ by 14 amino acids and possess distinct transcriptional capacities are present in cells due to alternative splicing of the CREBP mRNA. Aim 1 is to determine the significance of the dimerization of CREBP isoforms for transactivation and to establish whether the amount or distribution of isoforms is regulated. This will be done by examining the contribution of each peptide alone compared to the interactions of the same and different isoforms in a dimer and by determining whether CREBP synthesis or splicing is regulated by hormones. Aim 2 is to identify the functional domains of CREBP involved in activation of basal and cAMP- stimulated transcription. This will be done by creating specific mutations in the CREBP cDNA and by fusing potential activation domains to the GAL4 DNA-binding domain. The transactivational capacity of mutated CREBP proteins and of CREBP/GAL4 fusion factors will be assessed in transfection and in vitro transcription assays. Aim 3 is to identify and characterize the interactions of the CRE with heterologous promoter elements, especially those involved in regulation of PEPCK. This will identify potential targets of CREBP interactions. Domains of CREBP that are required for the interplay of different transcription factors can then be identified by examining the ability of different CREBP variants and fusion factors to potentiate activation by these other factors.

此处描述的研究计划的长期目标是了解营地如何特别强调基因表达营地响应元件（CRE）结合蛋白的作用机理（CREBP）和调节表达的因素相互作用磷酸烯醇丙酮酸羧基酶（PEPCK）基因。 CRE发挥着必不可少的在PEPCK基因的基础和cAMP刺激转录中的作用。 PEPCK的调节表达，催化速率限制步骤糖异生，对于维持适当的血糖至关重要浓度。基因转录受到相互作用的调节跨作用蛋白质因子彼此和RNA聚合酶，这是这些因素与顺式作用DNA元件的结合促进。循环 AMP激活蛋白激酶A，导致几种磷酸化蛋白质，包括CRE结合蛋白。 Crebp最近是纯化，克隆并显示直接参与基因调节表达。为了了解Crebp如何与其他因素相互作用以调节基因表达，有必要识别Crebp的域参与反式激活。 CREBP与CRE结合为二聚体，两个与14个氨基酸不同且具有独特转录的同工型由于CREBP的替代剪接，能力存在于细胞中 mRNA。目标1是确定CREBP二聚化的重要性进行反式激活的同工型，并确定金额还是同工型的分布受调节。这将通过检查与相同的相互作用相比，每个肽的贡献以及通过确定CREBP是否在二聚体中的不同同工型合成或剪接由激素调节。目标2是确定 CREBP的功能域参与基础和cAMP的激活刺激转录。这将通过创建特定的突变来完成在CREBP cDNA中并通过将电势激活结构融合到GAL4中 DNA结合域。突变Crebp的反式激活能力蛋白质和CREBP/GAL4融合因子将在转染中评估和体外转录测定。目标3是识别和表征 Cre与异源启动子元素的相互作用，尤其是参与PEPCK监管的人。这将确定潜力 CREBP相互作用的目标。 Crebp的域是所需的然后可以通过检查不同CREBP变体和融合因子的能力这些其他因素会增强激活。