| Summary: | 碩士 === 國立陽明大學 === 生命科學暨基因體科學研究所 === 96 === Chemokines are small, secreted proteins that play important roles such as regulating cell trafficking in homeostasis and inflammation in the immune system. Recently, a novel CXC chemokine-like protein, DMC (dendritic cell and monocyte chemokine-like protein), has been reported to specifically attract dendritic cells and monocytes. However, its structural nature and corresponding receptor remain unclear. Unlike the four-cysteine nature of CXC chemokines, DMC contains six cysteines. Conditioned media from DMC-transfected HEK-293T cells appear two major bands of DMC on SDS PAGE. Base on sequence alignment, two conserved convertase cutting sites between cysteine 2 and cysteine 3 were found. Mutagenesis was further applied to identify the correct cutting site for mature DMC production. We prepared 22 rat tissue cDNAs for real-time quantification of DMC expression and demonstrated DMC is highly expressed in the stomach. Further characterization showed that DMC is mainly transcribed from the glandular stomach. In the primary culture of epithelium cells from the glandular stomach, DMC expression levels were highly up-regulated in forskolin stimulation for 24 hrs, but were stable when stimulating with either gastrin or insulin. In the animal fasting experiments, DMC expression levels were stable in fasting and refeeding conditions. However, in the alcohol drinking experiment, DMC expression levels were up-regulated in the glandular stomach and lung but down-regulated in the forestomach. Immunohistochemical staining indicated the DMC protein is mainly localized on the mucosa layers of glandular stomach. Interestingly, a strong DMC staining was also found on the mucosa layer of forestomach, suggesting there might be DMC receptor expression. Whole genome GPCR analyses suggest there are six chemokines-like receptors remaining orphan. To pair DMC with these receptor candidates, the selected receptor genes were subsequently cloned. Furthermore, we also developed a novel chimeric reporting system that covers Gs, Gq, G12 and Gi pathways for future screening the ligand signaling of orphan GPCRs.
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