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 EP1、EP2、EP3和EP4介导前列腺素E_2(PGE_2)的作用。 EP3受体调节肾脏、胃的水和离子转运 胃酸分泌、神经递质释放和肝脏葡萄糖代谢。 在肾脏中，前列腺素E_2的产生调节盐和水的运输 髓质粗升支和集合管。重要的是，证据 提示EP3的某些效应是由基底外侧表面介导的。 肾单位，而其他的是由心尖受体介导的。《第三季》 受体在EP受体家族中是独一无二的，因为它以 共同前体的选择性剪接产生的多种异构体 从单个基因转录而来的mrna。尽管EP3受体具有 经典地被描述为GI偶联受体，通过 降低细胞内cAMP水平，最近的研究表明EP3 选择性剪接变异体通过多种信号转导途径耦合 小路。拟议的研究将重点放在大脑的功能差异上。 选择性剪接的PGE2受体亚型从而提供洞察力 探讨多种前列腺素E_2受体在正常和 病理生理状态。这项提议的主要假设是 EP3受体剪接变异体在个体中的差异表达 细胞类型导致信号通路的差异激活， 确定靶细胞对PGE2的生理反应。具体而言 目标#1首席调查员将确定曲目和组织 EP3受体剪接变异体信使RNA的分布。 将进行免疫定位，包括亚细胞受体 利用受体选择性靶向靶向EP3受体蛋白 相关组织和细胞培养模型中的抗体。在……里面 特异性靶向#2编码兔EP3受体剪接变异体的cDNA 将在哺乳动物细胞系中表达，并与其配体结合和 表征了信号转导特性。第三个具体目标是 确定特定位点氨基酸替换对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EP3剪接变体，也会增加我们的理解 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.