Cloning, expression, and functional analysis of human dopamine D1 receptors
文献类型：期刊论文
| 作者 | Sun, WC; Jin, L; Cao, Y; Wang, LZ; Meng, F; Zhu, XZ |
| 刊名 | ACTA PHARMACOLOGICA SINICA
 |
| 出版日期 | 2005-01 |
| 卷号 | 26期号:1页码:27-32 |
| 关键词 | cAMP response element-binding protein alkaline phosphatase reporter genes G-protein-coupled receptors dopamine D1 receptor radioligand assay calcium fluorescence screening |
| ISSN号 | 1671-4083 |
| DOI | 10.1111/j.1745-7254.2005.00017.x |
| 文献子类 | Article |
| 英文摘要 | Aim: To construct an HEK293 cell line stably expressing human dopamine D-1 receptor (D1R). Methods: cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned D1R cDNA was sequenced and stably expressed in HEK293 cells. Expression of D1R in HEK293 cells was monitored by the [H-3]SCH23390 binding assay. The function of D1R was studied by the cAMP accumulation assay, CRE-SEAP reporter gene activity assay, and intracellular calcium assay. Results: An HEK293 cell line stably expressing human D1R was obtained. A saturation radioligand binding experiment with [H-3]SCH23390 demonstrated that the K-d and B-max values were 1.5 +/- 0.2 nmol/L and 2.94 +/- 0.15 nmol/g of protein, respectively. In the [3H]SCH23390 competition assay, D1R agonist SKF38393 displaced [H-3]SCH23390 with an IC50 value of 2.0 (1.5-2.8) mumol/L. SKF38393 increased the intracellular cAMP level and CRE-SEAP activity through D1R expressed in HEK293 cells in a concentration-dependent manner with an EC50 value of 0.25 (0.12-0.53) mumol/L and 0.39 (0.27-0.57) mumol/L at 6 h/0.59 (0.22-1.58) mumol/L at 12 h, respectively. SKF38393 also increased the intracellular calcium level in a concentration-dependent manner with EC50 value of 27 (8.6-70) nmol/L. Conclusion: An HEK293 cell line stably expressing human D1R was obtained successfuly. The study also demonstrated that the CRE-SEAP activity assay could be substituted for the cAMP accumulation assay for measuring increase in cAMP levels. Thus, both intracellular calcium measurements and the CRE-SEAP activity assay are suitable for high-throughput screening in drug research. |
| WOS关键词 | PROTEIN-COUPLED RECEPTORS ; CULTURE ; SYSTEM ; ASSAY ; CALCIUM |
| WOS研究方向 | Chemistry ; Pharmacology & Pharmacy |
| 语种 | 英语 |
| CSCD记录号 | CSCD:480004 |
| WOS记录号 | WOS:000226426600004 |
| 出版者 | ACTA PHARMACOLOGICA SINICA |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/273946]  |
| 专题 | 药理学第二研究室 |
| 通讯作者 | Zhu, XZ |
| 作者单位 | 1.Chinese Acad Sci, Shanghai Inst Biol Sci, Inst Mat Med, Dept Pharmacol, Shanghai 201203, Peoples R China 2.Univ Michigan, Dept Psychiat, Psychiat MHRI Microarray Lab, Ann Arbor, MI 48109 USA |
| 推荐引用方式 GB/T 7714 | Sun, WC,Jin, L,Cao, Y,et al. Cloning, expression, and functional analysis of human dopamine D1 receptors[J]. ACTA PHARMACOLOGICA SINICA,2005,26(1):27-32. |
| APA | Sun, WC,Jin, L,Cao, Y,Wang, LZ,Meng, F,&Zhu, XZ.(2005).Cloning, expression, and functional analysis of human dopamine D1 receptors.ACTA PHARMACOLOGICA SINICA,26(1),27-32. |
| MLA | Sun, WC,et al."Cloning, expression, and functional analysis of human dopamine D1 receptors".ACTA PHARMACOLOGICA SINICA 26.1(2005):27-32. |
入库方式： OAI收割
来源：上海药物研究所
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