Functional Analysis of the Prostaglandin EP3 Receptor

描述(申请人提供)：前列腺素E2(PGE2)是一种主要的 花生四烯酸的环氧合酶代谢物和一种有效的广谱调节剂 各种细胞反应，包括血管张力，也包括发热反应 随着水和离子在肾脏中的运输。现在有确凿的证据表明，大多数 前列腺素E_2的S效应是通过特定的鸟嘌呤核苷酸调节来实现的 蛋白偶联受体命名为EP1、EP2、EP3和EP4。EP3受体 在EP受体家族中是唯一的，因为它由 通过选择性剪接产生的多个选择性剪接变体 共同前体基因的单个基因。这其中的主要假设是 提出了EP3受体剪接变异体在细胞上的差异表达 不同的细胞类型导致信号通路的不同激活 决定靶细胞对PGE2的生理反应。隐含在 这一假设是指EP3受体变体在功能上 不同的组织分布、细胞表达和细胞内 以及不同的信号转导特性。至 检验这一假设，在特定的目标1，我们将确定生理作用 使用转基因小鼠策略对EP3受体剪接变异体的研究 表达受限制的EP3受体剪接变异体。我们会 表征以血压和血管为重点的表型变化 反应性。尽管EP3受体被经典地描述为 GI偶联受体，通过降低细胞内cAMP水平传递信号，最近 研究表明EP3选择性剪接变异体通过其他 非GI信号转导通路。在具体目标2中，研究将侧重于 EP3受体介导的cAMP非依赖性机制的研究 信号转导及其对基因转录的影响。我们将研究 这一途径可能通过特定的基因调控基因表达 蛋白印迹分析中的抑制物和磷酸化特异性抗体， 启动子分析。

DESCRIPTION (provided by applicant): Prostaglandin E2 (PGE2) is a major cyclooxygenase metabolite of arachidonic acid and a potent modulator of a wide variety of cellular responses including vascular tone, febrile response as well as water and ion transport in the kidney. There is now firm evidence that most of PGE2's effects are mediated via specific guanine nucleotide regulatory protein coupled receptors designated EP1, EP2, EP3, and EP4. The EP3 receptor is the unique among the EP receptor family in that it is represented by multiple alternatively spliced variants generated by alternative splicing from a single gene of a common precursor mRNA. The principal hypothesis of this proposal is that differential expression of EP3 receptor splice variants on individual cell types results in differential activation of signaling pathways that determine the physiologic response of a target cell to PGE2. Implicit in this hypothesis is the notion that the EP3 receptor variants have functionally distinct tissue distribution, cellular expression, and intracellular compartmentalization as well as distinct signal transduction properties. To test this hypothesis, in Specific Aim 1 we will determine the physiologic role of the EP3 receptor splice variants using an genetically modified mice strategy expressing a restricted repertoire of EP3 receptor splice variants. We will characterize phenotypic changes focusing on blood pressure, and vascular reactivity. 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 other non-Gi signal transduction pathways. In Specific Aim 2, studies will focus on the characterization of the EP3 receptor-mediated mechanism of cAMP independent signal transduction and its effect on gene transcription. We will examine the potential for this pathway to modulate gene expression using specific inhibitors and phosphorylation-specific antibodies in Western blot analysis, and promoter analysis.