多巴胺D_1受体Ala229Thr多态性对受体信号转导功能的影响

Influence of dopamine D_1 receptor Ala229Thr polymorphism on receptor signal transduction

目的:构建表达不同基因型的人多巴胺D_1受体(DRD_1)真核细胞表达载体,明确DRD_1Ala229Thr多态性对受体信号转导功能的影响。方法:从人基因组经PCR扩增DRD_1基因编码区全长,将纯化后的PCR产物与pMD_19-T载体进行T连接,得到DRD_1229Ala(野生型)重组克隆载体。在野生型重组克隆载体的基础上,应用定点突变方法构建229Thr(突变型)重组克隆载体。用KpnⅠ和EcoR Ⅰ双酶切野生型和突变型重组克隆载体,将酶切后得到的目的片段纯化后与经相同双酶切的pc DNA3.1(+)表达载体连接,即得到野生型和突变型DRD_1-pcDNA3.1(+)重组真核细胞表达载体。将pc DNA3.1(+)空载体、野生型和突变型DRD_1-pcDNA3.1(+)重组真核细胞表达载体瞬时转染入COS-7细胞,用c AMP ELISA试剂盒分别检测forskolin和不同浓度多巴胺及(±)-SKF38393处理下细胞内基础cAMP的水平及激动剂诱导的cAMP的水平。结果:经双酶切及测序证实野生型和突变型DRD_1-pcDNA3.1(+)重组真核细胞表达载体构建正确。在forskolin处理的情况下,空载体组和阴性对照组细胞内cAMP的水平分别为(0.40±0.14)和(0.78±0.24)pmol/well,两者之间并没有显著差异(P=0.082),野生型组和突变型组细胞内c AMP的水平分别为(2.06±0.35)和(1.37±0.12)pmol/well,野生型组细胞内c AMP水平显著高于空载体组(P<0.001)和突变型组(P=0.007);在多巴胺及SKF38393处理的情况下,细胞内cAMP的水平均呈浓度依赖性升高,但突变型组细胞内的cAMP水平均显著低于野生型组(P<0.001)。多巴胺的EC_(50)值在野生型组为625 nmol/L,突变型组为9 612 nmol/L,比野生型组增加15倍;SKF38393的EC_(50)值在野生型组为421 nmol/L,突变型组为417 nmol/L,两者之间不存在明显差异。结论:本研究成功构建了野生型和突变型DRD_1-pcDNA3.1(+)重组真核细胞表达载体;DRD_1Ala229Thr多态性影响受体的激活和信号转导功能,与野生型相比,229Thr型受体信号转导功能显著降低。

AIM： To construct the eukaryotic expression vector carrying human dopamine D1 receptor （ DRDj ） gene in order to determine the influence of Ala229Thr polymorphism on the receptor signal transduction. METHODS： Site-directed muta- genesis and gene recombination techniques were used to construct the recombinant eukaryotic expression vectors containing different genotypes of DRD, gene. The coding sequence of DRD1 gene was cloned from human genome DNA by poIymerase chain reaction （PCR）. Purified PCR product was ligated to pMD19-T vector. Mutant DRD,-pMD19-T vector by site-directed mutagenesis was established based on wild DRDI-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 eukaryotic expression vectors, pcDNA3. 1 （ ＋ ） empty vector, wild and mutant DRDI-pcDNA3. 1 （ ＋ ） vectors were transiently transfected into COS-7 cells. 48 h after transfection, short-term exposure of cells to 100/zmol/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 re- ceptor （MT） group were （2.06 ± 0.35 ） and （ 1.37 ±0.12 ） pmolfwell, 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 ECs0 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 EC50 value for SKF38393 at the WT group was 421 nmol/L and at the MT group was 417 nmol/L, which was similar to that obtained at the WT group. CONCLUSION： DRDI-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.