Functional analysis of the D-2L 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 D-2 dopamine receptor. Saturation binding analysis using [H-3]spiperone showed that the receptor was expressed at low levels (B-max = 96.5 +/- 15.8 fmol/mg), but with an affinity characteristic of the D-2 receptor (K-d = 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 > (+) butaclarnol > 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 D-2 dopamine receptors, which are negatively coupled to the adenylyl cyclase signaling pathway, when heterogously expressed at low levels in CHO cells.