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

发布时间：2018-05-31 06:16

本文选题：食欲素受体 + 胆囊收缩素受体　；参考：《中国生物化学与分子生物学报》2017年08期

【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(P0.05)。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。
[Abstract]:Orexin 1 receptor (OX1R) and cholecystokinin receptor (CCK2R) were highly expressed in colon cancer cells, and the abnormal expression of OX1R- CCK2R was closely related to the proliferation of colon cancer cells induced by its ligand, but the specific mechanism was not clear. Previous studies have confirmed that OX1R and CCK1R can act as dimers in HT-29 cells. In this paper, a variety of (fluorescence) resonance energy transfer techniques (fret) combined with immunoprecipitation Co-IPP were used to further study the interaction between OX1R and CCK2R in living cells. The results of Bioluminescence Energy Resonance transfer (BRET) showed that when the amount of OX1R-Rluca was controlled and the amount of CCK2R-e YFPtransfected was increased gradually, it was compared with that of unstimulated (control) cells. After stimulation of orexin or gastrin, the BRET signal of HEK293T cells increased with the increase of receptor expression at 5 min, and reached the maximum value. Fluorescence resonance energy transfer technique was used to detect the FRET signals of OX1R-e YFP and CCK2Re CFP in HEK293T cells. At the same time, the results of receptor bleaching FRET(ap fret revealed that the receptor protein OX1R-e YFP-induced complete photobleaching of specific regions of the cell membrane expressing OX1R-e YFP and CCK2R-e CFP at the same time, which destroyed the receptor-donor interaction and energy transfer. The fluorescence intensity of donor CCK2R-e CFP was significantly higher than that before bleaching, and the fluorescence resonance energy transfer efficiency (FREP) of CCK2R-e CFP was significantly increased, which was 3.7 times as much as that of control transfected cells (P0.05). In addition, the results of gene transfection combined with Co-IP showed that HA-OX1Rnc-CCK2R fusion protein could be detected at the same time in the immunoprecipitation of HEK293T cell extracts cotransfected with Myc-CCK2R, while in the untransfected or single-transfected Myc-CCK2R cell extracts, HA-OX1Rnc-CCK2R fusion protein could be detected at the same time. However, two fusion proteins could not be detected at the same time. These results suggest that OX1R can interact with CCK2R under living cell physiological conditions, which provides a new clue to further study the role of OX1R in colon cancer cell proliferation and related signal pathways.
【作者单位】：济宁医学院神经生物研究所;
【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)~~
【分类号】：R735.35

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