DEVELOPMENT OF A DUAL GLOW-SIGNAL FIREFLY AND RENILLA LUCIFERASE ASSAY REAGENT FOR THE ANALYSIS OF G-PROTEIN COUPLED RECEPTOR SIGNALLING

AbstractSeveral reporter gene assays have been described where gene transcription is activated as a consequence of a specific signal transduction event, such as activation of adenylyl cyclase (1,2). Reporter genes typically consist of 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 assay for the simultaneous analysis of agonist activity at two G-protein coupled receptors which signal through activation of the G-protein alpha sub-unit, Gαs. Chinese hamster ovary (CHO) cells stably transfected with a cAMP responsive firefly luciferase reporter were further transfected with the human Vasopressin V2 receptor. Similarly, CHO cells stably transfected with a cAMP responsive Ren...