Development of a dual glow-signal firefly and Renilla luciferase assay reagent for the analysis of G-protein coupled receptor signalling

Several reporter gene assays have been described where gene transcription i s activated as a consequence of a specific signal transduction event, such as activation of adenylyl cyclase (1,2). Reporter genes typically consist o f specific responsive elements placed upstream of a minimal promoter, which together control the expression of a readily detectable reporter protein, such as luciferase. We have developed a dual glow-signal firefly and Renilla luciferase assay, which allows the simultaneous measurement of two reporter genes in the same well of a 96-well plate. In this report we demonstrate the use of this ass ay for the simultaneous analysis of agonist activity at two G-protein coupl ed receptors which signal through activation of the G-protein alpha sub-uni t, G alpha(s). Chinese hamster ovary (CHO) cells stably transfected with a cAMP responsive firefly luciferase reporter were further transfected with t he human Vasopressin V-2 receptor. Similarly, CHO cells stably transfected with a cAMP responsive Renilla luciferase reporter were further transfected with the human beta(2)-adrenoceptor. The two cell lines were mixed in indi vidual wells of a 96-well plate and a number of compounds were screened to determine their activity at both receptors. Stimulation with vasopressin an d beta(2)-adrenoceptor agonists resulted in the activation of the firefly a nd Renilla luciferases respectively. Stimulation with forskolin, which dire ctly stimulates adenylyl cyclase, caused the activation of both reporter ge nes, and stimulation with a range of further compounds with no activity at either receptor did not generate a reporter response. The dual luciferase a ssay allows the simultaneous screening of two receptors in a 96-well format resulting in significant time and cost savings.