Functional Analysis of the D 2L Dopamine Receptor Expressed in a cAMP-Responsive Luciferase Reporter Cell Line

• Published in: Biochemical pharmacology Vol. 56; no. 1; pp. 25 - 30
• Main Authors: George, Samantha E, Bungay, Peter J, Naylor, Louise H
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01-07-1998
• Subjects: adenylyl cyclase; CHO cells; CRE response element; dopamine (D 2) receptor; luciferase; reporter gene assay; adenylyl cyclase; luciferase; dopamine (D 2) receptor; reporter gene assay; CHO cells; CRE response element
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/S0006-2952(98)00014-8

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 [
3H]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 > (+)-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 D
2 dopamine receptors, which are negatively coupled to the adenylyl cyclase signaling pathway, when heterogously expressed at low levels in CHO cells.