PAF Receptor Trafficking and Function in Epithelial Cells

描述（由申请人提供）：血小板活化因子（PAF）是克罗恩病、溃疡性结肠炎和新生儿坏死性小肠结肠炎（NEC）等胃肠道疾病发病机制中的关键介质。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PAFR的运输和信号转导仅在非上皮组织中进行了研究。我们的前期研究表明PAF在实验性NEC中起着至关重要的作用，PAFR仅定位于培养的肠上皮细胞的顶端质膜，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。本提案中提供的初步数据表明，破坏抗洗涤剂膜结构域（DRM）、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。多不饱和脂肪酸是重要的营养素，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是关键的介导者。我们假设PAFR通过其胞质尾区C317的棕榈酰化作用靶向DRM，并且PUFA对棕榈酰化和DRM靶向作用的破坏可以抑制PAFR的信号转导效率。将基于以下三个具体目的来检验该假设：1）鉴定在极化上皮细胞的顶端质膜中将PAFR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来调节信号传导。3)研究PAFR棕榈酰化和PUFA对PAFR靶向和体内实验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，在新生大鼠肠道使用腺病毒基因转移和转基因小鼠沿着DRM和棕榈酰化的药理学操作。将使用成像、免疫学和生物化学方法分析PAFR的靶向，并使用高度可重复的功能测定评价功能。这些研究将阐明PAFR在上皮细胞中的靶向机制，以及PUFA可能影响PAFR和其他GPCR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.