REGULATION OF THE RAT VIP RECEPTOR GENE

大鼠 VIP 受体基因的调控

• 批准号: 6024442
• 负责人: LIN PEI
• 金额: $ 7.57万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6024442
• 关键词: Escherichia coli; expression cloning; fusion gene; gene expression; genetic library; genetically modified animals; hormone receptor; laboratory mouse; phosphorylation; protein sequence; receptor binding; receptor expression; recombinant proteins; vasoactive intestinal peptide

Vasoactive intestinal peptide (VIP) is a 28 amino acid polypeptide with a broad spectrum of biological functions. The effects of VIP are mediated by high affinity receptors. cDNA cloning of the VIP receptor showed that it is a G-protein coupled receptor containing seven transmembrane domains, and that it is expressed in a variety of tissues. The long term objective of this proposal is to understand the mechanisms that regulates VIP receptor gene expression during development and in disease. The Specific Aims in the original KO8 Award (DK02346) were: (1) To isolate and characterize the rat VIP receptor gene. (2) To identify cis-acting elements and DNA binding proteins important for transcriptional regulation of the VIP receptor gene in lung cells. (3) To study regulation of VIP receptor expression during rat development. (4) To study regulation of VIP receptor expression in carcinoma cell lines. The goals for the current proposal are to carry out those studies in the specific aims list above that have not been accomplished in the first three years of the KO8 Award. The Specific Aims are: (1) To map the DNA binding and trans-repession domains of VIPR-RP, and determined the functional importance of the phosphorylation sites. To map the DNA binding domain of VIPR-RP, different regions of the protein will be expressed in and purified from E.coli. The recombinant protein will be use in gel mobility shift assays with the VIPR-RP binding site as the probe. The trans-repression domain will be determined by making fusion constructs containing various regions of the VIPR-RP and the Gal- 4 DNA binding domain. The transcription repression activity of each construct will be tested by co-transfecting Cos7 cells with a reporter plasmid containing binding sites for Gal4 and luciferase. The functional importance of each putative sites for protein kinases within the DNA binding or trans-repression domain will be determined by in vitro kinase assays, and mutagenesis. (2) To generate DNA constructs that will be used for targeted disruption of the VIPR1 gene. A mouse genomic library will be screened using the rat VIPR1 gene as a probe to isolate the mouse VIPR1 gene. A Cre-loxP recombination approach will be used. A targeting vector containing two lox sites, and a vector containing a cell type-specifically expressed Cre transgene will be generated. These DNA constructs will be used in the future to generate transgenic mice that contain either null mutation or cell type-specific disruption of the VIPR1 gene. These studies will provide important insight into the physiological functions of VIP and the molecular mechanisms that regulates VIP receptor expression.

血管活性肠肽（VIP）是一种由 28 个氨基酸组成的多肽，具有广泛的生物学功能。 VIP 的作用由高亲和力受体介导。 VIP受体的cDNA克隆表明它是一种含有7个跨膜结构域的G蛋白偶联受体，并且在多种组织中表达。 该提案的长期目标是了解发育和疾病期间调节 VIP 受体基因表达的机制。最初的 KO8 奖 (DK02346) 的具体目标是： (1) 分离和表征大鼠 VIP 受体基因。 (2) 鉴定对肺细胞中VIP受体基因转录调节重要的顺式作用元件和DNA结合蛋白。 (3)研究大鼠发育过程中VIP受体表达的调控。 (4)研究癌细胞系中VIP受体表达的调控。当前提案的目标是开展上述具体目标中 KO8 奖前三年尚未完成的研究。 具体目标是： (1) 绘制 VIPR-RP 的 DNA 结合和反式复制结构域图谱，并确定磷酸化位点的功能重要性。 为了绘制 VIPR-RP 的 DNA 结合域图谱，该蛋白的不同区域将在大肠杆菌中表达并纯化。 重组蛋白将用于以 VIPR-RP 结合位点作为探针的凝胶迁移率变化测定。反式阻抑结构域将通过制备含有VIPR-RP和Gal-4 DNA结合结构域的各个区域的融合构建体来确定。 通过用含有 Gal4 和荧光素酶结合位点的报告质粒共转染 Cos7 细胞来测试每个构建体的转录抑制活性。 DNA 结合或反式抑制域内每个蛋白激酶推定位点的功能重要性将通过体外激酶测定和诱变来确定。 (2) 生成用于靶向破坏 VIPR1 基因的 DNA 构建体。 将使用大鼠VIPR1基因作为探针来筛选小鼠基因组文库以分离小鼠VIPR1基因。 将使用 Cre-loxP 重组方法。 将生成包含两个 lox 位点的靶向载体和包含细胞类型特异性表达的 Cre 转基因的载体。 这些 DNA 构建体将来将用于生成含有 VIPR1 基因无效突变或细胞类型特异性破坏的转基因小鼠。这些研究将为VIP的生理功能和调节VIP受体表达的分子机制提供重要的见解。