FUNCTION ANALYSIS OF THE PROSTAGLANDIN EP3 RECEPTOR

描述（改编自申请人的摘要）：分子克隆具有 鉴定了至少四种E-前列腺素（EP）受体亚型，命名为 EP 1、EP 2、EP 3和EP 4介导前列腺素E2（PGE 2）的作用。 EP 3受体调节肾脏、胃和胃中的水和离子转运。 酸分泌、神经递质释放和肝葡萄糖代谢。 在肾脏中，PGE 2的产生调节了盐和水在肾脏中的转运。 髓质粗的升支和集合管。 重要的是，证据 表明一些EP 3效应是由基底外侧表面介导的。 肾单位，而另一些是由顶端受体介导的。 EP3 受体在EP受体家族中是独特的，因为它作为 通过共同前体的选择性剪接产生的多种同种型 从单个基因转录的mRNA。 虽然EP 3受体 典型地被表征为Gi偶联受体，其通过 降低细胞内cAMP水平，最近的研究表明，EP 3 选择性剪接变异体通过多种信号转导偶联 途径。 拟议的研究将集中在功能差异， 选择性剪接的PGE 2受体亚型，从而提供了解 多种PGE 2受体在正常和 病理生理状态。 这一建议的主要假设是 EP 3受体剪接变体在个体中差异表达 细胞类型导致信号通路的差异激活， 测定靶细胞对PGE 2的生理反应。 在特定 目标#1主要研究者将确定剧目和组织 EP 3受体剪接变体信使RNA的分布。 将进行免疫定位，包括亚细胞受体 使用受体选择性靶向选择的EP 3受体蛋白 相关组织以及细胞培养模型中的抗体。 在 具体目标#2选择的编码兔EP 3受体剪接变体的cDNA 将在哺乳动物细胞系中表达， 信号转导特性表征。 第三个具体目标将 确定位点特异性氨基酸取代对G蛋白的影响 激活受体磷酸化和受体脱敏。 这些研究的完成不仅将阐明 这些多个EP 3剪接变异体，但也将增加我们的理解， G蛋白偶联受体的结构和功能。

DESCRIPTION (Adapted from the Applicant's Abstract): Molecular cloning has identified at least four E-Prostanoid (EP) receptor subtypes, designated EP1, EP2, EP3 and EP4 which mediate the effects of Prostaglandin E2 (PGE2). The EP3 receptor modulates water and ion transport in the kidney, gastric acid secretion, neurotransmitter release, and hepatic glucose metabolism. In the kidney PGE2 production modulates salt and water transport in the medullary thick ascending limb and collecting duct. Importantly, evidence suggests that some EP3 effects are mediated from the basolateral surface of the nephron, while others are mediated by apical receptors. The EP3 receptor is unique among the EP receptor family in that it exists as multiple isoforms generated by alternative splicing of a common precursor mRNA transcribed from a single gene. Although the EP3 receptor has classically been characterized as a Gi coupled receptor that signals by lowering intracellular cAMP levels, recent studies suggest that EP3 alternative splice variants couple through a variety of signal transduction pathways. The proposed studies will focus on the functional differences of the alternatively spliced PGE2 receptor subtypes thereby providing insight into the roles of the multiple PGE2 receptors in normal and pathophysiological states. The principal hypothesis of this proposal is that differential expression of EP3 receptor splice variants in individual cell types results in differential activation of signaling pathways that determine the physiologic response of a target cell to PGE2. In Specific Aim #1 the principal investigator will determine the repertoire and tissue distribution of EP3 receptor splice variant messenger RNAs. Immuno-localization will be performed, including subcellular receptor targeting of selected EP3 receptor proteins using receptor selective antibodies in relevant tissues as well as in cell culture models. In Specific Aim #2 selected cDNAs encoding rabbit EP3 receptor splice variants will be expressed in mammalian cell lines and their ligand binding and signal transduction properties characterized. The third Specific Aim will determine the effects of site-specific amino acid substitutions on G-protein activation receptor phosphorylation and receptor desensitization. Completion of these studies will not only elucidate the significance of these multiple EP3 splice variants, but will also increase our understanding of the structure and function of G-protein coupled receptors.