Use of a Dual Firefly and Renilla Luciferase Reporter Gene Assay to Simultaneously Determine Drug Selectivity at Human Corticotrophin Releasing Hormone 1 and 2 Receptors
✍ Scribed by S.J.W. Parsons; S.A. Rhodes; H.E. Connor; S. Rees; J. Brown; H. Giles
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 74 KB
- Volume
- 281
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
The aim of this study was to investigate and validate the use of a dual glow-signal luciferase reporter gene assay to simultaneously evaluate drug activity at two different seven-transmembrane receptor subtypes.
Stable cell lines (CHO
) transfected with either human corticotrophin releasing hormone 1 (hCRH 1 ) receptors and a firefly luciferase reporter gene or hCRH 2 and a Renilla luciferase reporter gene were created to provide different luciferase readouts for CRH 1 and CRH 2 receptors, respectively. Cells were combined for stimulation and measurement of luciferase luminescence in a 96-well plate format assay. The nonselective CRH agonists rat/human CRH and sauvagine caused concentration-dependent increases in luminescence via activation of CRH 1 (firefly luciferase; pEC 50 ؍ 8.40 ؎ 0.06 and 8.39 ؎ 0.08, respectively, n ؍ 8) and CRH 2 (Renilla luciferase; pEC 50 ؍ 8.89 ؎ 0.14 and 8.92 ؎ 0.13, respectively, n ؍ 8) receptors. The nonselective CRH antagonist astressin blocked these agonist-induced increases in luciferase at both CRH 1 and CRH 2 receptors. The selective CRH 1 antagonist CP154,526 blocked r/hCRH-and sauvagine-induced increases in luciferase at CRH 1 receptors only. These data report the expected pharmacology for CRH 1 and CRH 2 receptors. This assay enabled two receptor subtypes to be studied simultaneously in the same 96-well plate and generated robust data with low variability. It has the potential advantage of significant time and cost savings, with application to both basic research and compound screening.