人源受体hGPCRc工程细胞株的建立及其应用

Establishment and application of the engineered cells expressing a human G-protein-coupled receptor c

目的建立孤儿G蛋白偶联受体(OrphanGproteincoupledreceptors,oGPCRs)配基筛选体系并应用于化合物的筛选。方法利用RTPCR从人结肠组织获得oGPCR成员hGPCRc的氨基酸编码序列,在利用相关软件对hGPCRc的结构特点进行分析的基础上,构建hGPCRc之表达载体pcDNA3.1(+)hGPCRc,转染CHOK1细胞获得CHOhGPCR工程细胞株,将不同化合物作用于细胞株,用Fluo3为分子探针检测细胞内钙离子浓度变化,以分析化合物中是否为该受体的特异性配基。结果生物信息学分析得到:hGPCR定位于人染色体13q32.2,对应的氨基酸序列组成了7个跨膜区段的结构域,在进化树上与人源P2Y1受体最亲近,应属于人类GPCR成员;成功得到CHOhGPCRc工程细胞株;所检测化合物作用于细胞株并没有引起胞内钙离子的波动,很可能没有活化hGPCRc。结论hGPCRc是一个与已知人P2Y1最近的成员,但基于CHOhGPCRc工程细胞株的第二信使筛选结果表明,hGPCRc并不被P2Y1的已知配基活化因此很可能属于不同于P2Y1的嘌呤类受体新亚型。

Aim To establish a screening system of orphan G protein-coupled receptors (oGPCRs) for their ligands based on monitoring [Ca 2+]_i in engineered cells. Methods The whole ORF of a member of human oGPCR, designated human G-protein-coupled receptor c (hGPCRc), was amplified by RT-PCR from human colon tissue and its structure was analyzed with softwares. CHO-K_1 cells were transfected with the recombinant pcDNA 3.1(+)-hGPCRc to obtain engineered CHO-hGPCRc cells. As fluorescence probe, Fluo-3 was used in assaying the [Ca 2+]_i changes induced by different compounds in the CHO- hGPCRc cells.Results Bioinformatic analysis showed that hGPCRc was localized at 13q32.2, and its corresponding amino acids formed seven-transmembrane domains and was close to human P2Y_1 receptor. It was indicated that hGPCRc was a new member of human GPCR. CHO- hGPCRc cells expressing hGPCRc were obtained successfully but no one was able to activate hGPCRc among the tested compounds indicated by the [Ca 2+]_i changes. Conclusion Although hGPCRc was even though close to human P2Y_1 receptor, it can not be activated by the known compounds which activate the P2Y_1 receptor. hGPCRc might be a new member of purine receptor family but dose not belong to P2Y_1 subfamily.