中国临床药理学与治疗学2017，Vol.22Issue(10)：1106-1111,6.
多巴胺D1受体Ala229Thr多态性对受体信号转导功能的影响
Influence of dopamine D1 receptor Ala229Thr polymorphism on receptor signal transduction
彭娟 1黎翠林 2刘剑秋 2李智2
作者信息
- 1. 江西省人民医院药学部,南昌330006,江西
- 2. 中南大学湘雅医院临床药理研究所,长沙410008,湖南
- 折叠
摘要
Abstract
AIM:To construct the eukaryotic expression vector carrying human dopamine D1 receptor (DRD1) gene in order to determine the influence of Ala229Thr polymorphism on the receptor signal transduction.METHODS:Site-directed mutagenesis and gene recombination techniques were used to construct the recombinant eukaryotic expression vectors containing different genotypes of DRD1 gene.The coding sequence of DRD1 gene was cloned from human genome DNA by polymerase chain reaction (PCR).Purified PCR product was ligated to pMD19-T vector.Mutant DRD1-pMD19-T vector by site-directed mutagenesis was established based on wild DRD1-pMD19-T vector.After using Kpn Ⅰ and EcoR Ⅰ double endonucleases to excise DRD1-pMD19-T vector and pcDNA3.1 (+) empty vecor,both wild and mutant DRD1 gene CDS were then ligated to pcDNA3.1 (+) vectors to construct wild and mutant DRD1-pcDNA3.1 (+) recombinant eukary.otic expression vectors.pcDNA3.1 (+)empty vector,wild and mutant DRD1-pcDNA3.1 (+) vectors were transiently transfected into COS-7 cells.48 h after transfection,short-term exposure of cells to 100 μmol/L forskolin,dopamine and (±)-SKF38393 with various concentrations,intracellular cAMP accumulations were measured by enzymelinked immunosorbent assay (ELISA).RESULTS:DRD1-pcDNA3.1 (+) recombinant eukaryotic expression vectors were verified correctly by enzyme digestion as well as sequence analysis.The cAMP accumulations induced by 100μmol/L forskolin were (0.40 ± 0.14) pmol/well for pcDNA3.1 (+) empty vector group and (0.78 ± 0.24) pmol/well for mock-transfected group (P =0.082).The cAMP accumulations in response to forskolin treatment for the wild-type receptor (WT) group and mutant receptor (MT) group were (2.06 ±0.35) and (1.37± 0.12) pmol/well,respectively.The WT group presented markedly higher cAMP accumulations than pcDNA3.1 (+) empty vector group (P ＜ 0.001)and the MT group (P =0.007).The agonist-induced cAMP accumulations both increased in a concentration-dependent manner in COS-7 cells transfected WT and MT.Moreover,the MT group had a remarkable reduction in agonist-induced cAMP accumulations compared with the WT group (all P ＜0.001 for dopamine and SKF38393).In addition,intracellular cAMP maximal accumulation and EC50 value were investigated to evaluate the influence of MT on the efficacy and potency of agonists.Our data demonstrated that agonist-induced intracellular cAMP maximal accumulations of the MT group were significantly lower than those of the WT group (dopamine,P ＜ 0.001;SKF38393,P ＜ 0.001).The EC50 value for dopamine at the WT group was 625 nmol/L and at the MT group was 9612 nmol/L,a 15-fold increase over that obtained at the WT group.However,the ECs0 value for SKF38393 at the WT group was 421 nmol/L and at the MT group was 417nmol/L,which was similar to that obtained at the WT group.CONCLUSION:DRD1-pcDNA3.1(+) recombinant eukaryotic expression vectors were constructed successfully;Ala229Thr polymorphism affect the activation and signal transduction of DRD1,with the 229Thr receptor exert a lower signal transduction effect than wild type receptor.关键词
多巴胺D1受体/Ala229Thr多态性/信号转导/cAMPKey words
dopamine D1 receptor (DRD1)/Ala229Thr polymorphism/signal transduction/cAMP分类
医药卫生引用本文复制引用
彭娟,黎翠林,刘剑秋,李智..多巴胺D1受体Ala229Thr多态性对受体信号转导功能的影响[J].中国临床药理学与治疗学,2017,22(10):1106-1111,6.
