PAF Receptor Trafficking and Function in Epithelial Cells

描述（由申请人提供）：血小板活化因子（PAF）已被认为是胃肠道疾病如克罗恩病、溃疡性结肠炎和新生儿坏死性小肠结肠炎（NEC）发病机制的关键介质。PAF受体（PAFR）是G蛋白偶联受体（GPCR）超家族的成员。尽管PAF在胃肠道疾病中的重要性，以及PAFR在肠上皮细胞中的高水平表达，但目前仅在非上皮组织中研究了PAFR的转运和信号转导。我们之前的研究表明，PAF在实验性NEC中起着至关重要的作用，在培养的肠上皮细胞中，PAFR仅定位于顶质膜，并调节基因表达、离子转运、细胞内pH和细胞死亡等多种细胞功能。该提案中提供的初步数据表明，洗涤剂抗性膜结构域（DRM）的破坏，阻断棕榈酰化，或用多不饱和脂肪酸（PUFA）处理肠上皮细胞可消除或减弱paf诱导的细胞反应。多聚脂肪酸是一种重要的营养物质，在发育、健康和疾病方面具有广泛的生物学效应。特别是，PUFA在NEC的实验模型中发挥预防作用，其中PAF是一个关键的介质。我们假设PAFR通过其细胞质尾部C317的棕榈酰化靶向DRM，并且PUFA破坏棕榈酰化和DRM靶向可以抑制PAFR信号转导的效率。我们将基于以下三个具体目标来验证这一假设：1)确定PAFR在极化上皮细胞顶质膜上靶向DRM的机制，并确定DRM靶向在PAFR信号传导中的重要性。2)确定PUFA是否可以通过影响PAFR靶向DRM来调节信号。3)研究PAFR棕榈酰化和PUFA对PAFR靶向和体内实验性NEC的影响。为了实现这些目标，我们将利用异源表达、标记的野生型和突变型PAFR在极化上皮细胞系中，利用腺病毒基因转移在新生大鼠肠道中，以及在转基因小鼠中使用DRM和棕榈酰化的药理学操作。将使用影像学、免疫学和生化方法分析PAFR的靶向性，并使用高度可重复的功能分析来评估功能。这些研究将阐明PAFR靶向上皮细胞的机制，以及PUFA可能影响PAFR和其他GPCR-s功能的新机制。

DESCRIPTION (provided by applicant): Platelet-activating factor (PAF) has been implicated as a key mediator in the pathogenesis of gastrointestinal diseases such as Crohn's disease, ulcerative colitis and neonatal necrotizing enterocolitis (NEC). The PAF receptor (PAFR) is a member of the G protein-coupled receptor (GPCR) superfamily. Despite the importance of PAF in gastrointestinal diseases, and the high level expression of PAFR in intestinal epithelial cells, PAFR trafficking and signal transduction have been studied only in non-epithelial tissues. Our previous studies have shown that PAF plays a crucial role in experimental NEC, PAFR is localized exclusively in the apical plasma membrane in cultured intestinal epithelial cells and regulates such diverse cellular functions as gene expression, ion transport, intracellular pH, and cell death. Preliminary data presented in this proposal shows that disruption of detergent resistant membrane domains (DRM), blocking palmitoylation, or treatment of intestinal epithelial cells with polyunsaturated fatty acids (PUFA) eliminates, or blunts PAF-induced cellular responses. PUFA are important nutrients, with a broad range of biological effects in development, health and disease. In particular, PUFA play a preventive role in an experimental model of NEC, where PAF is a critical mediator. We hypothesize that PAFR is targeted to DRM via palmitoylation of C317 in its cytoplasmic tail, and that disruption of palmitoylation and DRM targeting by PUFA can inhibit the efficiency of signal transduction by PAFR. This hypothesis will be tested based on the following three specific aims: 1) To identify the mechanisms that target PAFR to DRM in the apical plasma membrane of polarized epithelial cells and to determine the importance of DRM targeting in PAFR signaling. 2) To determine whether PUFA can modulate signaling by affecting PAFR targeting to DRM. 3) To examine the effect of PAFR palmitoylation and PUFA on PAFR targeting and on experimental NEC in vivo. In order to accomplish these goals we will utilize heterologously expressed, tagged wild type and mutant PAFR in polarized epithelial cell lines, in neonatal rat intestine using adenoviral gene transfer and in transgenic mice along with the pharmacological manipulation of DRM and palmitoylation. Targeting of PAFR will be analyzed using imaging, immunologic and biochemical methods, and function will be evaluated using highly reproducible functional assays. These studies will elucidate the mechanisms of PAFR targeting in epithelial cells, and a novel mechanism by which PUFA might affect the function of PAFR, and other GPCR-s.