Expression of dopamine D_1 receptor in Sf9 insect cells and agonism of 1-12-chloroscoulerine on recombinant D_1 receptor  

多巴胺D_1受体在Sf9昆虫细胞中的表达及左旋氯代斯阔任对重组D_1受体的激动作用(英文)

作　　者：和友[1] 金文桥[1] 申庆祥 陈新建 金国章 

机构地区：[1]中国科学院上海生命科学研究院药物研究所,上海200031 [2]上海市计划生育研究所

出　　处：《Acta Pharmacologica Sinica》2003年第3期225-229,共5页中国药理学报（英文版）

基　　金：Project supported by the National Natural Science Foundation of China (№ 30070866) and Grant G (1998) 051126 from the Ministry of Science and Technology of China.

摘　　要：AIM: To express dopamine D_1 receptor in baculovirus-Sf9 cell system, and to investigate the effects of l-12-chloroscoulerine (l-CSL) on the recombinant D_1 receptor (D_1R). METHODS: The recombinant baculovirus, Autographa californica nuclear polyhedrosis virus bearing D_1R (AcNPV- D_1R) was generated, and then was used to produce recombinant D_1R in Sf9 insect cells. Expression of D_1R in Sf9 cells was monitored by [3~H]SCH23390 binding assay. The effects of l-CSL on recombinant D_1R were investigated by [3~H]SCH23390 binding assay and cAMP assay. RESULTS: The recombinant baculovirus AcNPV bearing D_1R cDNA was generated, and was successfully expressed in Sf9 insect cells. The expression level of (B_(max)) was (0.94±0.06) nmol/g protein. The K_d value of [3~H]SCH23390 was (1.9±0.3) nmol/L, which was consistent with the previous results from calf striutam tissues. l-CSL had a high affinity to recombinant D_1R with K_i value of (6.3±1.4) nmol/L, and increased the intracellular cAMP level in a concentration-dependent manner with EC_(50) value of 0.72 μmol/L and 95％ confidence limit was 0.67-0.77 μmol/L. Thus l-CSL has the D_1 receptor agonism. CONCLUSION: An efficient baculovirus Sf9 insect cell system for dopamine D_1 receptor was constructed and l-CSL presented the D_1 receptor agonism on cellular-molecular level directly.目的：在Sf9昆虫细胞中表达D1受体，并研究左旋氯代斯阔任对重组D1受体的激动作用。方法：构建含D1受体cDNA的重组杆状病毒，以其感染Sf9昆虫细胞得到D1受体表达。[^3H]SCH23390受体结合检测重组D1受体的药理特性，[^3H]SCH23390受体结合和cAMP测定实验检测左旋氯代斯阔任对重组D1受体的激动作用。结果：在Sf9昆虫细胞中成功表达D1受体，[^3H]SCH23390与重组D1受体最大结合量（Bmax）为（0.94±0.06)nmol/g蛋白，[^3H]SCH23390与重组D1受体的结合解离常数（Kd）为（1.9±0.3)nmol/L，其药理特性与小牛纹状体脑匀浆所得结果一致，左旋氯代斯阔任对重组D1受体有高亲和力，解离常数Ki为（6.3±1.4)nmol/L；并剂量依赖地引起胞内cAMP增加，EC50为0.72μmol/L(95%可信限为0.67-0.77μmol/L)，表现出D1激动作用。结论：在杆状病毒/昆虫细胞Sf9中，成功建立了D1受体异源表达系统。在细胞分子水平，直接证实了左旋氯代斯阔任对D1受体的激动作用。

关 键 词：recombinant proteins dopamine D_1 receptors INSECTS BACULOVIRIDAE l-12-chloroscoulerine cyclic AMP 

分 类 号：R962[医药卫生—药理学]