Regulation of Vascular Eicosanoid Receptors

描述(申请人提供)：前列环素(PG I2)和血栓素A2(TXA2)是血管系统中形成的两种主要环氧合酶产物，具有相反的生物学效应。PGI2是一种有效的抑制血小板聚集、血管扩张剂、抗血栓和抗有丝分裂原的药物，在血管疾病中可能具有保护作用。相反，诱导血小板聚集、有丝分裂和血管收缩的TXA2似乎在心血管疾病的发病机制中起着有害的作用。PGI2和TXA2都是通过分别与它们的细胞表面受体IP和TP(两个剪接变体α和β)结合来转导它们的作用。IP和TP经常在心血管细胞中共表达，在血管疾病中PGI2和TXA2的生物合成增加是一致的。在平滑肌细胞、血小板和其他类型的细胞中，PGI2和TXA2反应事件的交叉调节是明显的。然而，尽管我们了解了这两个系统所利用的调节和信号转导途径，但对每个信号级联对另一个系统的影响知之甚少。这项建议旨在研究人类(H)IP和HTP单独和协同激活是否以及如何相互调节，并试图了解伴随这些相互作用的分子机制。我们将解决以下具体假设1.HIP和HTP的共同激活调节了它们的调节和G蛋白偶联。我们将研究HIP和hTPalpha/β在内源性表达两种受体类型的血管平滑肌细胞中的异源调节，或缺乏其中一种受体，以及在单独或联合表达受体的细胞系中。激动剂诱导IP和TPalpha/β的同型和异型二聚化发生，并具有功能和调节后果。我们将研究人血管平滑肌细胞、IP或TP缺乏小鼠的培养细胞以及表达HIP/HTP的细胞系中二聚体的形成和受体信号转导。髋关节和髋关节与新的细胞蛋白质相互作用。CGMP-磷酸二酯酶-6增量亚单位，一种新的HIP相关因子，在HIP信号和调节中的作用将被研究。我们将使用酵母双杂交技术来筛选血管平滑肌细胞的cDNA文库，并用蛋白质组学的方法来研究其他与HIP或hTP相互作用的未知蛋白。这些研究将在受体信号和调控水平上定义两个血管活性二十烷类化合物的相互作用，并确定G蛋白偶联受体，特别是IP和TP，可以异源调控受体功能的新途径。

DESCRIPTION (provided by applicant): Prostacyclin (PG I2) and thromboxane (TxA2), two major cyclooxygenase products formed in the vasculature, exert opposing biological effects. PGI2 is a potent inhibitor of platelet aggregation, vasodilator, anti-thrombotic and anti-mitogen which may be protective in settings of vascular disease. In contrast, TxA2, which induces platelet aggregation, mitogenesis and vasoconstriction, appears to play a detrimental role in the pathogenesis of cardiovascular disorders. Both PGI2 and TxA2 transduce their effects by binding to their cell surface receptors, IP and TP (two splice variants alpha and beta), respectively. IP and TP are frequently co-expressed in cardiovascular cells and elevated PGI2 and TxA2 biosynthesis is coincident in vascular disorders. Cross-regulation of PGI2 and TxA2-responsive events is evident in smooth muscle cells, platelets and other cells types. However, despite our understanding of the regulation and signal transduction pathways utilized by these two systems, little is known about the impact of each signaling cascade on the other. This proposal aims to examine whether and how human (h) IP and hTP activation alone and in concert may modulate each other and seeks to understand the molecular mechanisms attendant to these interactions. We shall address following specific hypotheses.1. That co-activation of hIP and hTP modulates their regulation and G protein coupling. We shall examine heterologous regulation of hIP and hTPalpha/beta in vascular smooth muscle cells endogenously express both receptor types, or deficient in one or the other, and in cell lines expressing the receptors alone or in combination.2. That agonist-induced homo- and hetero- dimerization of IP and TPalpha/beta occurs and has functional and regulatory consequences. We shall examine dimer formation and receptor signaling in vascular smooth muscle cells from humans or cultured from IP or TP deficient mice and in hIP/hTP expressing cell lines.3. That hIP and hIP interact with novel cellular proteins. The role of the cGMP-phosphodiesterase-6 delta subunit, a novel hIP associated factor, in hIP signaling and regulation will be investigated. We shall use yeast-two hybrid technology to screen a vascular smooth muscle cell cDNA library and a proteomic approach to investigate other uncharacterized proteins that interact with hIP or hTP.These studies will define the interaction of two vasoactive eicosanoids at the level of receptor signaling and regulation and identify novel pathways through which G protein-coupled receptors in general, and IP and TP in particular, can heterologously modulate receptor function.