cAMP Biosensor Assay Using BacMam Expression System: Studying the Downstream Signaling of LH/hCG Receptor Activation
| Autor: | Sergei Kopanchuk, Tõnis Laasfeld, Ago Rinken, Maris-Johanna Tahk, Darja Lavogina, Anni Allikalt, Olga Kukk |
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| Rok vydání: | 2021 |
| Předmět: |
0303 health sciences
Chemistry BacMam Peptide hormone Cell biology 03 medical and health sciences chemistry.chemical_compound Transduction (genetics) 0302 clinical medicine Förster resonance energy transfer Second messenger system Cyclic adenosine monophosphate Receptor 030217 neurology & neurosurgery Plate reader 030304 developmental biology |
| Zdroj: | Methods in Molecular Biology ISBN: 9781071612200 |
| DOI: | 10.1007/978-1-0716-1221-7_12 |
| Popis: | 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 Forster-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. |
| Databáze: | OpenAIRE |
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