Expression of dopamine D1 receptor in Sf9 insect cells and agonism of l-12-chloroscoulerine on recombinant D1 receptor.
OBJECTIVE
To express dopamine D1 receptor in baculovirus-Sf9 cell system, and to investigate the effects of l-12-chloroscoulerine (l-CSL) on the recombinant D1 receptor (D1R).


METHODS
The recombinant baculovirus, Autographa californica nuclear polyhedrosis virus bearing D1R (AcNPV- D1R) was generated, and then was used to produce recombinant D1R in Sf9 insect cells. Expression of D1R in Sf9 cells was monitored by [3H]SCH23390 binding assay. The effects of l-CSL on recombinant D1R were investigated by [3H]SCH23390 binding assay and cAMP assay.


RESULTS
The recombinant baculovirus AcNPV bearing D1R cDNA was generated, and was successfully expressed in Sf9 insect cells. The expression level of (Bmax) was (0.94+/-0.06) nmol/g protein. The Kd value of [3H]SCH23390 was (1.9+/-0.3) nmol/L, which was consistent with the previous results from calf striatum tissues. l-CSL had a high affinity to recombinant D1R with Ki value of (6.3+/-1.4) nmol/L, and increased the intracellular cAMP level in a concentration-dependent manner with EC50 value of 0.72 micromol/L and 95 % confidence limit was 0.67-0.77 micromol/L. Thus l-CSL has the D1 receptor agonism.


CONCLUSIONS
An efficient baculovirus-Sf9 insect cell system for dopamine D1 receptor was constructed and l-CSL presented the D1 receptor agonism on cellular-molecular level directly.
He
You
Y
Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200031.
Jin
Wen-Qiao
WQ
Shen
Qing-Xiang
QX
Chen
Xin-Jian
XJ
Jin
Guo-Zhang
GZ
D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001600:Berberine Alkaloids / Q000494:pharmacology*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017447:Receptors, Dopamine D1 / Q000819:agonists / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism / Q000821:virology*; D013237:Stereoisomerism; D014162:Transfection
C099468:12-chloroscoulerine; D001600:Berberine Alkaloids; D017447:Receptors, Dopamine D1; D011994:Recombinant Proteins; D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D000242:Cyclic AMP