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



To construct an HEK293 cell line stably expressing human dopamine D1 receptor (D1R).


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.


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.


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.


  1. 1

    Goldman-Rakic PS . The cortical dopamine system: role in memory and cognition. Adv Pharmacol 1998; 42: 707–11.

    CAS  Article  Google Scholar 

  2. 2

    Schultz W . Getting formal with dopamine and reward. Neuron 2002; 36: 241–63.

    CAS  Article  Google Scholar 

  3. 3

    Yelnik J . Functional anatomy of the basal ganglia. Mov Disord 2002; 17: Suppl 3: S15–S21.

    Article  Google Scholar 

  4. 4

    Nicola SM, Surmeier J, Malenka RC . Dopaminergic modulation of neuronal excitability in the striatum and nucleus accumbens. Annu Rev Neurosci 2000; 23: 185–215.

    CAS  Article  Google Scholar 

  5. 5

    Missale C, Nash SR, Robinson SW, Jaber M, Caron MG . Dopamine receptors: from structure to function. Physiol Rev 1998; 78: 189–225.

    CAS  Article  Google Scholar 

  6. 6

    Zhou QY, Grandy DK, Thambi L, Kushner JA, van Tol HH, Cone R, et al. Cloning and expression of human and rat D1 dopamine receptors. Nature 1990; 347: 76–80.

    CAS  Article  Google Scholar 

  7. 7

    Knight PJ, Pfeifer TA, Grigliatti TA . A functional assay for G-protein-coupled receptors using stably transformed insect tissue culture cell lines. Anal Biochem 2003; 320: 88–103.

    CAS  Article  Google Scholar 

  8. 8

    Okun I, Veerapandian P . New methods to mimic nature in high-throughput screening. Nat Biotechnol 1997; 15: 287–8.

    CAS  Article  Google Scholar 

  9. 9

    Dove A . Drug screening — beyond the bottleneck. Nat Biotechnol 1999; 7: 859–63.

    Article  Google Scholar 

  10. 10

    Sorensen K, Brodbeck U . Assessment of coating-efficiency in ELISA plates by direct protein determination. J Immunol Methods 1986; 95: 291–3.

    CAS  Article  Google Scholar 

  11. 11

    Nordstedt C, Fredholm BB . A modification of a protein-binding method for rapid quantification of cAMP in cell-culture supernatants and body fluid. Anal Biochem 1990; 189: 231–4.

    CAS  Article  Google Scholar 

  12. 12

    Durocher Y, Perret S, Thibaudeau E, Gaumond MH, Kamen A, Stocco R, et al. A reporter gene assay for high-throughput screening of Gprotein-coupled receptors stably or transiently expressed in HEK293 EBNA cells grown in suspension culture. Anal Biochem 2000; 284: 316–26.

    CAS  Article  Google Scholar 

  13. 13

    Kassack MU, Hofgen B, Lehmann J, Eckstein N, Quillan JM, Sadee W . Functional screening of G protein-coupled receptors by measuring intracellular calcium with a fluorescence microplate reader. J Biomol Screen 2002; 7: 233–46.

    CAS  PubMed  Google Scholar 

  14. 14

    Johnson RA, Salomon Y . Assay of adenylyl cyclase catalytic activity. Methods Enzymol 1991; 195: 3–21.

    CAS  Article  Google Scholar 

  15. 15

    Chen W, Shields TS, Stork PJ, Cone RD . A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways. Anal Biochem 1995; 226: 349–54.

    CAS  Article  Google Scholar 

  16. 16

    Stratowa C, Himmler A, Czernilofsky AP . Use of a luciferase reporter system for characterizing G-protein-linked receptors. Curr Opin Biotechnol 1995, 6: 574–81.

    CAS  Article  Google Scholar 

  17. 17

    Pieroni JP, Jacobowitz O, Chen JQ, Lyengar R . Signal recognition and integration by GS-stimulated adenylyl cyclases. Curr Opin Neurobiol 1993; 3: 345–51.

    CAS  Article  Google Scholar 

  18. 18

    Hescheler J, Schultz G . G-proteins involved in the calcium channel signalling system. Curr Opin Neurobiol 1993; 3: 360–7.

    CAS  Article  Google Scholar 

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Corresponding author

Correspondence to Xing-zu Zhu.

Additional information

Project supported by the National Natural Science Foundation of China (No 30128004) and Shanghai Metropolitan Fund for Research and Development (No 04DZ14005).

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Sun, Wc., Jin, L., Cao, Y. et al. Cloning, expression, and functional analysis of human dopamine D1 receptors. Acta Pharmacol Sin 26, 27–32 (2005).

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

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