Expression of dopamine D₁ receptor in Sf9 insect cells and agonism of 1-12-chloroscoulerine on recombinant D₁ receptor  

多巴胺D1受体在Sf9昆虫细胞中的表达及左旋氯代斯阔任对重组D1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 [2]上海市计划生育研究所

出　　处：《Acta Pharmacologica Sinica》2003年第3期35-39,共页中国药理学报（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 D₁ receptor in baculovirus-Sf9 cell system, and to investigate the effects of l-12-chloroscoulerine （l-CSL） on the recombinant D₁ receptor （D₁R）. METHODS: The recombinant baculovirus, Autographa californica nuclear polyhedrosis virus bearing D₁R （AcNPV- D₁R） was generated, and then was used to produce recombinant D₁R in Sf9 insect cells. Expression of D₁R in Sf9 cells was monitored by [3^H]SCH23390 binding assay. The effects of l-CSL on recombinant D₁R were investigated by [3^H]SCH23390 binding assay and cAMP assay. RESULTS: The recombinant baculovirus AcNPV bearing D₁R 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 D₁R with K_i value of （6.3±1.4） nmol/L, and increased the intracellular cAMP level in a concentration-dependent manner with EC₅₀ value of 0.72 μmol/L and 95％ confidence limit was 0.67-0.77 μmol/L. Thus l-CSL has the D₁ receptor agonism. CONCLUSION: An efficient baculovirus Sf9 insect cell system for dopamine D₁ receptor was constructed and l-CSL presented the D₁ 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(B_max)为（0.94±0.06）nmol/g蛋白,[³H]SCH23390与重组D₁受体的结合解离常数（K_d）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K_i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 D₁ receptors INSECTS BACULOVIRIDAE l-12-chloroscoulerine cyclic AMP 

分 类 号：R96[医药卫生—药理学]