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 eukaryotic 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 enzyme-linked 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 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: 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.

Key words: dopamine D1 receptor (DRD1), Ala229Thr polymorphism, signal transduction, cAMP