Cloning, expression, and functional analysis of human dopamine D1 receptors.
OBJECTIVE
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 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) micromol/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) micromol/L and 0.39 (0.27-0.57) micromol/L at 6 h/0.59 (0.22-1.58) micromol/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.


CONCLUSIONS
An HEK293 cell line stably expressing human D1R was obtained successfully. 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.
Sun
Wan-chun
WC
Department of Pharmacology, Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Jin
Lei
L
Cao
Yan
Y
Wang
Li-zhen
LZ
Meng
Fan
F
Zhu
Xing-zu
XZ
D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000378:metabolism; D001552:Benzazepines / Q000494:pharmacology; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D018491:Dopamine Agonists / Q000494:pharmacology; D018492:Dopamine Antagonists / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D017447:Receptors, Dopamine D1 / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism
D001552:Benzazepines; D017362:Cyclic AMP Response Element-Binding Protein; D018491:Dopamine Agonists; D018492:Dopamine Antagonists; D007527:Isoenzymes; D017447:Receptors, Dopamine D1; D043562:Receptors, G-Protein-Coupled; C055791:germ-cell AP isoenzyme; D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D000242:Cyclic AMP; D000469:Alkaline Phosphatase; D002118:Calcium