Original Article

Cloning, expression, and functional analysis of human dopamine D1 receptors1

Wan-chun SUN, Lei JIN, Yan CAO, Li-zhen WANG, Fan MENG, Xing-zu ZHU


Aim: To construct an HEK293 cell line stably expressing human dopamine D1receptor (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 Kd and Bmax 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 activity
assay are suitable for high-throughput screening in drug research.