Regulation of Vascular Eicosanoid Receptors

描述（由申请方提供）：前列环素（PG I2）和血栓烷（TxA 2）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。PGI 2是一种有效的血小板聚集抑制剂、血管扩张剂、抗血栓形成剂和抗有丝分裂原剂，在血管疾病的情况下可能具有保护作用。与此相反，TxA 2，诱导血小板聚集，有丝分裂和血管收缩，似乎在心血管疾病的发病机制中发挥有害作用。PGI 2和TxA 2都通过分别与其细胞表面受体IP和TP（两种剪接变体α和β）结合来抑制其作用。IP和TP经常在心血管细胞中共表达，并且血管疾病中PGI 2和TxA 2生物合成的升高是一致的。PGI 2和TxA 2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的影响知之甚少。该建议旨在研究人类（h）IP和hTP激活是否以及如何单独和协同地相互调节，并试图了解这些相互作用的分子机制。我们将讨论以下具体假设。1. hIP和hTP的共激活调节它们的调节和G蛋白偶联。我们将研究hIP和hTPalpha/β在血管平滑肌细胞中的异源调节，所述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，以及在单独或组合表达受体的细胞系中。激动剂诱导的IP和TPa/β的同源和异源二聚化发生并具有功能和调节后果。我们将在来自人类或培养自IP或TP缺陷小鼠的血管平滑肌细胞中以及在hIP/hTP表达细胞系中检查二聚体形成和受体信号传导。hIP和hIP与新的细胞蛋白相互作用。cGMP-磷酸二酯酶-6 δ亚基，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。我们将利用酵母双杂交技术筛选血管平滑肌细胞cDNA文库，并利用蛋白质组学方法研究与hIP或hTP相互作用的其他未知蛋白，这些研究将在受体信号传导和调节水平上确定两种血管活性类花生酸的相互作用，并确定新的途径，通过这些途径，G蛋白偶联受体，特别是IP和TP，可以异源调节受体功能。

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.