食欲素1受体与胆囊收缩素2受体在细胞内的相互作用分析  

Analysis of the Interaction between Orexin Receptor 1 and Cholecystokinin Receptor 2 in Living Cells

作　　者：姜云璐 王正文[1] 程葆华[1] 白波[1] 陈京[1] JIANG Yun-Lu WANG Zheng-Wen CHENG Bao-Hua BAI Bo CHEN Jing(Neurobiology Institute, Jining Medical University, Jining 272067, Shandong, Chin)

机构地区：[1]济宁医学院神经生物研究所,山东济宁272000

出　　处：《中国生物化学与分子生物学报》2017年第8期789-798,共10页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.3097108;No.816712276;No.31271243;No.81070961);山东省自然科学基金项目(No.ZR2014HL404);山东省高校科技计划项目(No.JY2015KJ004;No.2015-57-6)~~

摘　　要：食欲素1受体(orexin 1 receptor,OX1R)与胆囊收缩素2受体(cholecystokinin receptor,CCK2R)在结肠癌细胞中高表达,且异常表达的OX1R、CCK2R与其配体诱导的结肠癌细胞增殖密切相关,但具体机制尚不清楚。以前的研究证实,OX1R与CCK1R在HT-29细胞中能以二聚体的形式发挥作用。本文利用多种(荧光)共振能量转移技术(FRET)结合免疫共沉淀(Co-IP),进一步研究活细胞中OX1R与CCK2R是否发生相互作用。生物发光能量共振转移(BRET)结果显示,在控制供体(OX1R-Rluc)量不变,而逐渐增加受体(CCK2R-e YFP)转染量时,与无刺激的(对照)细胞比较,食欲素或胃泌素刺激HEK293T细胞5 min,BRET信号伴随受体表达量的增加而增加,并达到最大值。采用荧光共振能量转移技术在HEK293T细胞中,能够检测到OX1R-e YFP与CCK2Re CFP明显的FRET信号。同时,受体漂白FRET(ap FRET)结果揭示,在同时表达OX1R-e YFP和CCK2R-e CFP的细胞膜特定区域,进行受体蛋白(OX1R-e YFP)完全光漂白、破坏了受体-供体之间的相互作用和能量传递后,由于供体(CCK2R-e CFP)荧光强度比漂白前明显增强,其荧光共振能量转移效率(FREPe)明显增加,是对照转染细胞的3.7倍(P<0.05)。此外,基因转染结合Co-IP结果显示,仅有在共转染HA-OX1R与Myc-CCK2R的HEK293T细胞提取液的免疫沉淀物中,可同时检出HA-OX1R、Myc-CCK2R融合蛋白,而在未转染或单转Myc-CCK2R的细胞提取液沉淀物中,却不能同时检出两种融合蛋白。以上结果表明,在活细胞生理条件下,OX1R可与CCK2R相互作用,这为进一步探讨二者相互作用在结肠癌细胞增殖中的作用及相关信号通路提供了新的线索。The orexin 1 receptor （OXIR） and cholecystokinin 2 receptor （CCK2R）, which are closely correlated to cellular proliferation, are highly expressed in colon cancer cells. Its mechanism, however, is unclear. Based on the previous finding that OXIR and CCK^R can form dimer in HT-29 cells, in this study we investigate whether OX1R and CCKzR may heterodimerize to function in living cells by using a variety of resonance energy transfer techniques and co-immunoprecipitation （Co-IP）. The bioluminescence resonance energy transfer （BRET） signals （BRET ratios） of OX1R and CCK2R in HEK293T cells were analyzed after the cells were exposed to orexin or gastrin for 5 minutes. Results showed that compared with non-exposed （control） cells, BRET signals were increased and reached a maximum when increased the acceptor （CCK2R-eYFP） expression without changes in donor （OXIR- Rluc） expression. The resonance energy transfer signals between OX1R-eYFP and CCK2R-eCFP over- expressed in HEK293T cells were detected by fluorescence resonance energy transfer （FRET） method. Moreover, receptor photobleaching FRET （apFRET） revealed that the FRET efficiency （FRETe） of the donor protein （CCK2R-eCFP） in the OXIR-eYFP and CCK2R-eCFP co-transfected living cells was significantly increased to 3.7 folds of that in transfected control cells （P 〈 0.05 ） after complete photobleaching which disrupted the receptor protein （OXIR-eYFP） and receptor-donor interaction. In addition, the combination of gene transfection and co-immunoprecipitation （Co-IP） indicated that the HA-OX1R and Myc-CCK2R fusion proteins were detectable only in the immunoprecipitates from HA- OX, R and Myc-CCK2R co-transfected cells but not in that from transfected control and/or Myc-CCK2R- transfected cells. These dates suggest that OX1R can interact with CCKzR in living cells, which may provide new clues for further investigating the role of OXIR-CCK1R interaction in colon cancer cell proliferation and the relative signaling pat

关 键 词：食欲素受体 胆囊收缩素受体 荧光共振能量转移 免疫共沉淀 

分 类 号：Q7[生物学—分子生物学]