MOLECULAR STUDIES OF RENAL DOPAMINE 1 RECEPTORS

肾脏多巴胺能系统活性降低与 高血压的发展和/或维持。外源性给药 或内源性多巴胺(DA)增加钠和水 排泄，主要通过刺激 肾脏D1R。大量证据表明，肾脏D1 受体主要集中在肾近端小管上，而D1R 刺激抑制近端小管钠重吸收。我们有 最近克隆了猪D1a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D1a受体基因是第一个克隆的D1a受体 已知在肾脏细胞中同源表达的基因。什么都不知道， 然而，关于肾脏D1a受体是如何在 基因转录或翻译。因此，LLC-PK1细胞是一个理想的模型 研究肾脏D1a受体基因调控的系统 转录和翻译水平。有5个具体目标。我们 建议： 1.研究天然D1a受体基因的转录调控 通过将细胞暴露于多种已知因素而在LLC-PK1细胞中表达 影响基因转录并监测稳定状态的变化 D1a受体基因表达水平。 2.鉴定该基因5‘侧翼区的调控结构域 猪D1a受体基因的连续构建 截短型D1a受体5‘侧翼区--氯霉素 乙酰基转移酶(CAT)报告基因融合构建及检测 瞬时转染LLC-PK1细胞的转录活性 基础和刺激条件(Sp.目标1)。 3.表征D1a的正、负调制区 受体基因启动子通过DNase I足迹、凝胶迁移率移动和 超位移分析。 4.确定D1a受体细胞特异性转录的基础 比较D1a受体启动子-CAT的转录活性 在肾脏(LLC-PK1、HEK-293、COS-7和OK)和非 肾(NS20Y和NB41A3小鼠神经母细胞瘤)细胞 已知的表达D1a受体(LLC-PK1， NS20Y，OK)产生与D1a受体不同的结合模式 推动者。 5.确定D1a受体5‘前导序列的程度 翻译的多肽以及它们是否在调节中发挥作用 受体蛋白的翻译。 这些研究将为监管提供重要的新见解 肾D1a受体在转录水平上的生理意义 和翻译水平。

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.