PAF Receptor Trafficking and Function in Epithelial Cells

描述(申请人提供)：血小板激活因子(PAF)在克罗恩病、溃疡性结肠炎和新生儿坏死性小肠结肠炎(NEC)等胃肠道疾病的发病机制中起关键作用。PAF受体(PAFR)是G蛋白偶联受体超家族的成员。尽管PAF在胃肠道疾病中具有重要作用，而且PAFR在肠上皮细胞中高水平表达，但PAFR的转运和信号转导仅在非上皮组织中得到研究。我们以前的研究表明，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，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。该提案中提出的初步数据表明，破坏抗洗剂膜域(DRM)、阻断棕榈酰化或用多不饱和脂肪酸(PUFA)处理肠上皮细胞可以消除或钝化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、健康和疾病中具有广泛的生物学效应。特别是，多不饱和脂肪酸在NEC的实验模型中发挥着预防作用，其中PAF是关键的介体。我们推测，PAFR通过胞浆尾部C317的棕榈酰化作用靶向DRM，而PUFA破坏棕榈酰化和DRM靶向性可以抑制PAFR的信号转导效率。这一假说将基于以下三个具体目标进行验证：1)确定极化上皮细胞顶端质膜中以PAFR为靶点的DRM的机制，并确定DRM靶向在PAFR信号转导中的重要性。2)确定PUFA是否可以通过影响PAFR靶向DRM来调制信号。3)检测PAFR棕榈酰化和多不饱和脂肪酸对PAFR靶向性和体内实验性NEC的影响。为了实现这些目标，我们将利用极化上皮细胞系中异源表达的、标记的野生型和突变型PAFR，通过腺病毒基因转移在新生大鼠的肠道中以及在转基因小鼠中利用DRM和棕榈酰化的药理操作。PAFR的靶向性将使用成像、免疫学和生化方法进行分析，功能将使用高度重复性的功能分析进行评估。这些研究将阐明PAFR在上皮细胞中的靶向机制，以及多不饱和脂肪酸可能影响PAFR和其他GPCR功能的新机制-S。

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.