REGULATION OF THE RAT VIP RECEPTOR GENE

血管活性肠肽（VIP）是一种由28个氨基酸组成的具有广泛生物学功能的多肽。VIP的作用是由高亲和力受体介导的。对VIP受体的cDNA克隆表明，它是一个含有7个跨膜结构域的g蛋白偶联受体，并在多种组织中表达。这项建议的长期目标是了解在发育和疾病过程中调节VIP受体基因表达的机制。原KO8 Award （DK02346）的具体目的是：(1)分离和表征大鼠VIP受体基因。(2)鉴定肺细胞中VIP受体基因转录调控的重要顺式作用元件和DNA结合蛋白。(3)研究VIP受体在大鼠发育过程中的表达调控。(4)研究VIP受体在癌细胞中的表达调控。目前提案的目标是开展上述具体目标清单中在KO8奖的头三年未完成的研究。具体目的是：(1)绘制VIPR-RP的DNA结合和反式抑制结构域，确定磷酸化位点的功能重要性。为了绘制VIPR-RP的DNA结合区域，将在大肠杆菌中表达和纯化该蛋白的不同区域。重组蛋白将用于以VIPR-RP结合位点为探针的凝胶迁移转移试验。反式抑制结构域将通过制作包含VIPR-RP和Gal- 4 DNA结合结构域的不同区域的融合构建体来确定。每种结构的转录抑制活性将通过用含有Gal4和荧光素酶结合位点的报告质粒共转染Cos7细胞来测试。在DNA结合或反式抑制区域内，每个假定的蛋白激酶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。以大鼠VIPR1基因为探针，筛选小鼠基因组文库，分离小鼠VIPR1基因。将使用Cre-loxP重组方法。将生成含有两个lox位点的靶向载体和含有细胞类型特异性表达的Cre转基因的载体。这些DNA构建物将在未来用于产生含有无突变或细胞类型特异性破坏VIPR1基因的转基因小鼠。这些研究将为VIP的生理功能和调控VIP受体表达的分子机制提供重要的见解。

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.