Acta Pharmacologica Sinica 2005 January; 26 (1): 27-32; doi: 10.1111/j.1745-7254.2005.00017.x

Aim: To construct an HEK293 cell line stably expressing human dopamine D₁ 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 [³H]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 [³H]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 [³H]SCH23390 with an IC₅₀ value of 2.0 (1.5-2.8) µmol/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) µmol/L and 0.39 (0.27-0.57) µmol/L at 6 h/0.59 (0.22-1.58) µmol/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 acti-vity assay are suitable for high-throughput screening in drug research.

Keywords: cAMP response element-binding protein; alkaline phosphatase; reporter genes; G-protein-coupled receptors; dopamine D1 receptor; radioligand assay; calcium; fluorescence; screening