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MOLECULAR STUDIES OF RENAL DOPAMINE 1 RECEPTORS

肾多巴胺 1 受体的分子研究

基本信息

批准号：
2460017
负责人：
DENNIS P HEALY
金额：
$ 26.84万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-08-01 至 1998-07-31
项目状态：
已结题

项目摘要

Decreased activity of the renal dopaminergic system has been linked to the development and/or maintenance of hypertension. Exogenously administered or endogenously generated dopamine (DA) increases sodium and water excretion, actions that are mediated primarily through stimulation of renal D1 receptors. A great deal of evidence indicates that renal D1 receptors are concentrated on kidney proximal tubules and that D1 stimulation inhibits proximal tubule sodium reabsorption. We have recently cloned the porcine D1A receptor gene and have shown that it is expressed in the proximal tubule-like porcine renal epithelial cell line, LLC-PK1. The porcine D1A receptor gene is the first cloned D1A receptor gene known to be expressed homologously in renal cells. Nothing is known, however, about how the renal D1A receptor is regulated at the level of gene transcription or translation. LLC-PK1 cells are thus an ideal model system to study the regulation of the renal D1A receptor gene at both the transcriptional and translational levels. There are 5 Specific Aims. We propose to: 1. Study the regulation of transcription of the native D1A receptor gene expressed in LLC-PK1 cells by exposing cells to a variety of factors known to influence gene transcription and monitoring changes in the steady state levels of D1A receptor mRNA. 2. Characterize the regulatory domains of the 5' flanking region of the porcine D1A receptor gene by constructing a series of successively truncated D1A receptor 5' flanking region-chloramphenicol acetyltransferase (CAT) reporter gene fusion constructs and measuring transcriptional activity in transiently transfected LLC-PK1 cells under basal and stimulated conditions (Sp. Aim #1). 3. Characterize the positive and negative modulatory regions of the D1A receptor gene promoter by DNase I footprinting, gel mobility shift and supershift assays. 4. Determine the basis for cell-specific transcription of the D1A receptor gene by comparing transcriptional activity of D1A receptor promoter-CAT constructs expressed in renal (LLC-PK1, HEK-293, COS-7 and OK) and non- renal (NS20Y and NB41A3 mouse neuroblastoma) cells and by determining if nuclear extracts from cells known to express the D1A receptor (LLC-PK1, NS20Y, OK) yield different patterns of binding to the D1A receptor promoter. 5. Determine the extent to which D1A receptor 5' leader sequence polypeptides are translated and whether they play a role in regulating translation of the receptor protein. These studies will provide important new insights into the regulation of the physiologically significant kidney D1A receptor at the transcriptional and translational levels.

肾多巴胺能系统活性降低与发展和/或维持高血压。外源施用的或内源性产生的多巴胺（DA）增加钠和水排泄，主要通过刺激肾D1受体大量证据表明，肾D1 受体集中在肾近端小管上，刺激抑制近端小管钠重吸收。我们有最近克隆了猪D1 A受体基因，并表明它是在近端小管样猪肾上皮细胞系中表达， LLC-PK1. 猪D1 A受体基因是第一个克隆的D1 A受体已知在肾细胞中同源表达的基因。什么都不知道，然而，关于肾脏D1 A受体如何在基因转录或翻译。 LLC-PK 1细胞因此是理想的模型系统研究肾D1 A受体基因在两个细胞的调节，转录和翻译水平。有五个具体目标。我们提议： 1.研究天然D1 A受体基因的转录调控通过将细胞暴露于多种已知的因子，以影响基因转录并监测稳定状态下的变化 D1 A受体mRNA水平。 2.表征所述多核苷酸的5'侧翼区的调控结构域，猪D1 A受体基因，截短D1 A受体5'侧翼区-氯霉素乙酰转移酶（CAT）报告基因融合构建体和测量瞬时转染LLC-PK 1细胞的转录活性基础和刺激条件（Sp.目标#1）。 3.表征D1 A的正和负调节区域通过DNA酶I足迹法、凝胶迁移率改变和超位移测定。 4.确定D1 A受体细胞特异性转录的基础通过比较D1 A受体启动子-CAT的转录活性，表达于肾（LLC-PK 1、HEK-293、COS-7和OK）和非肾（非肾）中的构建体肾（NS 20 Y和NB 41 A3小鼠神经母细胞瘤）细胞，并通过确定来自已知表达D1 A受体的细胞的核提取物（LLC-PK 1， NS 20 Y，OK）产生与D1 A受体的不同结合模式启动子 5.确定D1 A受体5'前导序列多肽被翻译，以及它们是否在调节受体蛋白的翻译。这些研究将提供重要的新的见解，生理上重要的肾脏D1 A受体的转录和翻译水平。