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. ? ? ?
