REGULATION OF THE RAT VIP RECEPTOR GENE

血管活性肠肽(VIP)是由28个氨基酸组成的多肽，具有广泛的生物学功能。VIP的作用是由高亲和力受体介导的。对VIP受体的克隆表明，VIP受体是一种G蛋白偶联受体，含有7个跨膜区，在多种组织中表达。这项建议的长期目标是了解VIP受体基因在发育和疾病中表达的调节机制。最初的KO8奖(DK02346)的具体目的是：(1)分离和鉴定大鼠VIP受体基因。(2)鉴定对肺细胞VIP受体基因转录调控重要的顺式作用元件和DNA结合蛋白。(3)研究VIP受体在大鼠发育过程中的表达规律。(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那些在KO8奖的头三年中尚未实现的研究。具体目的是：(1)绘制VIPR-RP的DNA结合域和反式转运域的图谱，并确定磷酸化位点的功能重要性。为了定位VIPR-RP的DNA结合域，我们将在大肠杆菌中表达并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率变化分析。反式阻遏结构域将通过构建包含VIPR-RP和Gal-4DNA结合域的不同区域的融合构建体来确定。通过将含有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。蛋白激酶在DNA结合或反式抑制结构域中的每个假定位置的功能重要性将通过体外激酶分析和突变来确定。(2)产生用于靶向干扰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。一个包含两个lox位点的靶向载体和一个包含细胞类型特异性表达的Cre转基因基因的载体将被产生。这些DNA构建物将在未来用于产生含有VIPR1基因零突变或特定细胞类型破坏的转基因小鼠。这些研究将对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。

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.