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

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 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 activity
assay are suitable for high-throughput screening in drug research.