Cloning, expression, and functional analysis of human dopamine D1 receptors

Aim: To construct an HEK293 cell line stably expressing human dopamine D 1 receptor (D 1 R). 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 D 1 R cDNA was sequenced and stably expressed in HEK293 cells. Expression of D 1 R in HEK293 cells was monitored by the [ 3 H]SCH23390 binding assay. The function of D 1 R 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 D 1 R was obtained. A saturation radioligand binding experiment with [ 3 H]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 [ 3 H]SCH23390 competition assay, D 1 R agonist SKF38393 displaced [ 3 H]SCH23390 with an IC 50 value of 2.0 (1.5–2.8) μmol/L. SKF38393 increased the intracellular cAMP level and CRE‐SEAP activity through D 1 R expressed in HEK293 cells in a concentration‐dependent manner with an EC 50 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 EC 50 value of 27 (8.6–70) nmol/L. Conclusion: An HEK293 cell line stably expressing human D 1 R 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.