Abstract
Cyclic adenosine monophosphate (cAMP) serves as a second messenger for numerous G-protein-coupled receptors. Changes in cellular cAMP levels reflect the biological activity of various GPCR-specific agents, including protein hormones. cAMP biosensors based on detection of Förster-type resonance energy transfer (FRET) offer unique advantages including the ratiometric nature of measurement, adjustable affinity toward detected molecule, capability of monitoring kinetics of cAMP release, and compatibility with the multi-well format and fluorescence plate reader platforms. In this chapter, we introduce the optimized version of the previously reported method to achieve sufficient and reproducible level of cAMP biosensor protein expression with the means of BacMam transduction system. As a practical challenge, we address the applicability of the designed assay for screening of biological activity of human hormones, including human chorionic gonadotropin (hCG) bearing different posttranslational modifications.
Key words
- cAMP assay
- BacMam
- Baculovirus
- Epac-SH188
- FRET
- Protein hormone activity
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Acknowledgments
We thank Dr. Prema Narayan (Southern Illinois University) for LHCGR-MDCK cells, and Professor Kees Jalink group (The Netherlands Cancer Institute) for cAMP sensor plasmids. The work has been financed by the Estonian Ministry of Education and Science (PRG454, PSG230) and by Enterprise Estonia (grant EU48695).
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Lavogina, D. et al. (2021). cAMP Biosensor Assay Using BacMam Expression System: Studying the Downstream Signaling of LH/hCG Receptor Activation. In: Martins, S.A.M., Prazeres, D.M.F. (eds) G Protein-Coupled Receptor Screening Assays. Methods in Molecular Biology, vol 2268. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1221-7_12
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DOI: https://doi.org/10.1007/978-1-0716-1221-7_12
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