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 [ 3H]spiperone showed that the receptor was expressed at...
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| Published in: | Biochemical pharmacology Vol. 56; no. 1; pp. 25 - 30 |
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| Main Authors: | , , |
| Format: | Journal Article |
| Language: | English |
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Elsevier Inc
01-07-1998
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| Abstract | 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. |
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| AbstractList | 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. |
| Author | Naylor, Louise H George, Samantha E Bungay, Peter J |
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| ContentType | Journal Article |
| Copyright | 1998 Elsevier Science Inc. |
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| DOI | 10.1016/S0006-2952(98)00014-8 |
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| References | Seeman, Van Tol (BIB17) 1994; 15 Civelli, Bunzow, Grandy (BIB1) 1993; 32 Wood (BIB8) 1995; 6 Migeon, Nathanson (BIB10) 1994; 269 Harley, Middlemiss, Ragan (BIB20) 1995; 115 George, Bungay, Naylor (BIB13) 1997; 69 Lazareno, Birdsall (BIB15) 1993; 109 Steffey, Roberts, Frail, Kebabian, MacKenzie (BIB16) 1993; 46 Castellano, Liu, Monsma, Sibley, Kapatos, Chiodo (BIB6) 1993; 44 Montmayeur, Borrelli (BIB12) 1991; 88 Castro, Strange (BIB22) 1993; 60 Vallar, Muca, Magni, Albert, Bunzow, Meldolesi, Civelli (BIB4) 1990; 265 Kanterman, Mahan, Briley, Monsma, Sibley, Axelrod, Felder (BIB5) 1991; 39 Himmler, Stratowa, Czernilofsky (BIB11) 1993; 13 Castañón, Spevak (BIB9) 1994; 198 Weiss, Sebben, Garcia-Sainz, Bockaert (BIB19) 1985; 27 Stratowa, Himmler, Czernilofsky (BIB7) 1995; 6 Albert, Neve, Bunzow, Civelli (BIB3) 1990; 265 Onali, Olianas, Gessa (BIB18) 1985; 28 Woodward, Coley, Daniell, Naylor, Strange (BIB14) 1996; 66 George, Bungay, Naylor (BIB21) 1997; 2 Gingrich, Caron (BIB2) 1993; 16 |
| References_xml | – volume: 109 start-page: 1110 year: 1993 end-page: 1119 ident: BIB15 article-title: Estimation of competitive antagonist affinity from functional inhibition curves using the Gaddum, Schild and Cheng-Prusoff Equations publication-title: Br J Pharmacol contributor: fullname: Birdsall – volume: 6 start-page: 50 year: 1995 end-page: 58 ident: BIB8 article-title: Marker proteins for gene expression publication-title: Curr Opin Biotech contributor: fullname: Wood – volume: 60 start-page: 372 year: 1993 end-page: 375 ident: BIB22 article-title: Differences in the ligand binding properties of the short and long versions of the D publication-title: J Neurochem contributor: fullname: Strange – volume: 16 start-page: 299 year: 1993 end-page: 321 ident: BIB2 article-title: Recent advances in the molecular biology of dopamine receptors publication-title: Ann Rev Neurosci contributor: fullname: Caron – volume: 15 start-page: 264 year: 1994 end-page: 270 ident: BIB17 article-title: Dopamine receptor pharmacology publication-title: Trends Pharmacol Sci contributor: fullname: Van Tol – volume: 13 start-page: 79 year: 1993 end-page: 94 ident: BIB11 article-title: Functional testing of human D publication-title: J Receptor Res contributor: fullname: Czernilofsky – volume: 44 start-page: 649 year: 1993 end-page: 656 ident: BIB6 article-title: Transfected-D publication-title: Mol Pharmacol contributor: fullname: Chiodo – volume: 6 start-page: 574 year: 1995 end-page: 581 ident: BIB7 article-title: Use of a luciferase reporter system for characterising G-protein-linked receptors publication-title: Curr Opin Biotech contributor: fullname: Czernilofsky – volume: 198 start-page: 626 year: 1994 end-page: 631 ident: BIB9 article-title: Functional coupling of human adenosine receptors to a ligand-dependent reporter gene system publication-title: Biochem Biophys Res Comm contributor: fullname: Spevak – volume: 88 start-page: 3135 year: 1991 end-page: 3138 ident: BIB12 article-title: Transcription mediated by a cAMP-responsive promoter element is reduced upon activation of dopamine D publication-title: Proc Natl Acad Sci USA contributor: fullname: Borrelli – volume: 32 start-page: 281 year: 1993 end-page: 307 ident: BIB1 article-title: Molecular diversity of the dopamine receptors publication-title: Ann Rev Pharm Toxicol contributor: fullname: Grandy – volume: 27 start-page: 595 year: 1985 end-page: 599 ident: BIB19 article-title: D publication-title: Mol Pharmacol contributor: fullname: Bockaert – volume: 115 start-page: 1307 year: 1995 end-page: 1313 ident: BIB20 article-title: Relationship between inhibition of cyclic AMP production in Chinese hamster ovary cells expressing the rat D publication-title: Br J Pharmacol contributor: fullname: Ragan – volume: 2 start-page: 1 year: 1997 end-page: 6 ident: BIB21 article-title: Evaluation of a CRE-directed luciferase reporter gene assay as an alternative to measuring cAMP accumulation publication-title: J Biomolec Screen contributor: fullname: Naylor – volume: 69 start-page: 1278 year: 1997 end-page: 1285 ident: BIB13 article-title: Functional coupling of endogenous serotonin (5-HT publication-title: J Neurochem contributor: fullname: Naylor – volume: 66 start-page: 394 year: 1996 end-page: 402 ident: BIB14 article-title: Investigation of the role of conserved serine residues in the long form of the rat D publication-title: J Neurochem contributor: fullname: Strange – volume: 265 start-page: 2098 year: 1990 end-page: 2104 ident: BIB3 article-title: Coupling of a cloned rat dopamine-D publication-title: J Biol Chem contributor: fullname: Civelli – volume: 46 start-page: 747 year: 1993 end-page: 751 ident: BIB16 article-title: Further characterizaton of the D publication-title: Biochem Pharmacol contributor: fullname: MacKenzie – volume: 265 start-page: 10320 year: 1990 end-page: 10326 ident: BIB4 article-title: Differential coupling of dopaminergic receptors expressed in different cell types publication-title: J Biol Chem contributor: fullname: Civelli – volume: 269 start-page: 9767 year: 1994 end-page: 9778 ident: BIB10 article-title: Differential regulation of cAMP-mediated gene transcription by m1 and m4 muscarinic acetylcholine receptors publication-title: J Biol Chem contributor: fullname: Nathanson – volume: 28 start-page: 138 year: 1985 end-page: 145 ident: BIB18 article-title: Characterization of dopamine receptors mediating inhibition of adenylate cyclase activity in rat striatum publication-title: Mol Pharmacol contributor: fullname: Gessa – volume: 39 start-page: 364 year: 1991 end-page: 369 ident: BIB5 article-title: Transfected D publication-title: Mol Pharmacol contributor: fullname: Felder |
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| Title | Functional Analysis of the D 2L Dopamine Receptor Expressed in a cAMP-Responsive Luciferase Reporter Cell Line |
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