Functional analysis of the D2L dopamine receptor expressed in a cAMP-responsive luciferase reporter cell line.
A Chinese hamster ovary (CHO) cell line expressing the firefly luciferase gene under the control of six cAMP response elements (CREs) was stably transfected with the long form of the rat D2 dopamine receptor. Saturation binding analysis using [3H]spiperone showed that the receptor was expressed at low levels (Bmax = 96.5+/-15.8 fmol/mg), but with an affinity characteristic of the D2 receptor (Kd = 21.5+/-3.7 pM). Luciferase expression in this cell line was modified in a dose dependent manner with dopamine receptor agonists (N-propylapomorphine > apomorphine > quinpirole > dopamine) and antagonists (spiperone > (+)-butaclamol > D0710 > (-)-sulpiride > tiapride > remoxipride), according to their rank order of potency in binding and cAMP accumulation studies. Dopamine-mediated inhibition of forskolin-stimulated luciferase expression was pertussis toxin sensitive. This demonstrated the efficiency of the luciferase reporter gene assay for the functional testing of D2 dopamine receptors, which are negatively coupled to the adenylyl cyclase signaling pathway, when heterogously expressed at low levels in CHO cells.
George
S E
SE
Department of Biosciences, The University of Kent, Canterbury, UK.
Bungay
P J
PJ
Naylor
L H
LH
D037361:Adenylate Cyclase Toxin; D000818:Animals; D016466:CHO Cells; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism*; D018491:Dopamine Agonists / Q000494:pharmacology; D018492:Dopamine Antagonists / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics*; D037342:Pertussis Toxin; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000235:genetics* / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D018491:Dopamine Agonists; D018492:Dopamine Antagonists; D017448:Receptors, Dopamine D2; D010566:Virulence Factors, Bordetella; C113776:dopamine D2L receptor; D005576:Colforsin; D000242:Cyclic AMP; D008156:Luciferases; D037342:Pertussis Toxin