Abstract
AbstractCyclic adenosine 3’,5’-monophosphate is an important second messenger molecule that regulates many downstream signaling pathways in cells. Detection of cAMP levels relies on screenings of cell lysates or the use of genetically encoded biosensors for detection in living cells. Genetically encoded biosensors are, however, primarily used for bioimaging and rarely in high-throughput screenings of potential drug candidates. Here, we describe a quantitative fluorescence-based imaging method based on measurements of single living cells. We used a genetically encoded Epac149 biosensor to investigate cAMP production in living cells following ligand stimulation. The study revealed a dependence of the measured cAMP levels on the expression level of the biosensor in transiently transfected cells. While the biosensor maintained linearity of the signal at high expression levels, the linearity of the biosensor was lost at lower expression levels due to a deficit of the biosensor compared to the maximum possible production of cAMP in the cells. This problem was circumvented by establishment of a stable cell line with constitutive expression of the biosensor. We established dose response curves by stimulation with the β1-adrenergic receptor partial agonist denopamine and observed up to 1.48-fold difference in the cellular response as well as up to 4.27-fold difference in LogEC50 values between cells with insufficient and sufficient biosensor expression. Careful characterization and control of the biosensor expression is therefore important in order to conduct quantitative analysis of the cAMP production and it allows the use of genetically encoded biosensor to be applied in high-throughput screenings.
Publisher
Cold Spring Harbor Laboratory
Cited by
1 articles.
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