Regulation of Vascular Eicosanoid Receptors

描述（由申请人提供）：Prostacyclin （PG I2）和凝血素（TxA2）是两种主要的环加氧酶产物，在血管系统中形成，发挥相反的生物效应。PGI2是一种有效的血小板聚集抑制剂，血管舒张剂，抗血栓形成和抗丝裂原，可能在血管疾病的设置保护。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发挥了不利作用。PGI2和TxA2分别通过结合细胞表面受体IP和TP（两个剪接变体α和β）来转导它们的作用。IP和TP经常在心血管细胞中共表达，PGI2和TxA2的生物合成升高与血管疾病是一致的。PGI2和txa2反应事件的交叉调节在平滑肌细胞、血小板和其他细胞类型中是明显的。然而，尽管我们了解这两个系统的调控和信号转导途径，但对每个信号级联对另一个信号级联的影响知之甚少。本提案旨在研究人类IP和http激活是否以及如何相互调节，并试图理解这些相互作用的分子机制。我们将讨论以下具体假设。hIP和hTP的共同激活调节了它们的调节和G蛋白偶联。我们将研究血管平滑肌细胞内源性表达两种受体类型，或缺乏一种或另一种受体，以及单独表达或组合表达受体的细胞系中hIP和hTPalpha/ β的异源调节。这种激动剂诱导的IP和TPalpha/ β的同源和异二聚化发生并具有功能和调节作用。我们将在人类血管平滑肌细胞、IP或TP缺陷小鼠培养的血管平滑肌细胞以及表达hIP/ TP的细胞系中检测二聚体的形成和受体信号。hIP和hIP与新的细胞蛋白相互作用。cgmp -磷酸二酯酶-6 δ亚基是一种新的hIP相关因子，在hIP信号传导和调节中的作用将被研究。我们将使用酵母- 2杂交技术筛选血管平滑肌细胞cDNA文库，并使用蛋白质组学方法研究与hIP或hTP相互作用的其他未表征的蛋白质。这些研究将确定两种血管活性类二十烷在受体信号传导和调控水平上的相互作用，并确定G蛋白偶联受体，特别是IP和TP，可以异源调节受体功能的新途径。

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.