BacMam system for FRET-based cAMP sensor expression in studies of melanocortin MC1 receptor activation.
Cyclic adenosine monophosphate (cAMP) is a second messenger of many G-protein-coupled receptors (GPCRs) and a useful readout molecule to estimate the biological activity of various GPCR-specific agents. Here we report the development and use of a Förster resonance energy transfer (FRET) biosensor for cAMP (Epac2-camps) combined with a baculovirus-based BacMam transduction system. The constructed BacMam-Epac2-camps viral transduction system is a simple and robust tool for ligand screening at the second-messenger level in a variety of mammalian cell lines. The level of biosensor protein expression can easily be adjusted in a dose-dependent manner depending on the multiplicity of viral infection. For setting up the assay, we used a B16F10 murine melanoma cell line with endogenous expression of melanocortin-1 receptor (MC(1)R). The receptor activation was characterized by a set of MC(1)R full and partial agonists. Bivalent ions Ca(2+) as well as Mg(2+) modulated ligand potencies, whereas the effect was ligand and ion specific. Results obtained for MC(1)R indicate that the BacMam-Epac2-camps system may also be applicable for studying the activation of other GPCRs and may be implemented in routine analysis as well as in high-throughput screening.
University of Tartu, Institute of Chemistry, Tartu, Estonia.
Olga
O
Mazina
Reet
R
Reinart-Okugbeni
Sergei
S
Kopanchuk
Ago
A
Rinken
D000818:Animals; D016367:Baculoviridae; D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000032:analysis* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D044102:Receptor, Melanocortin, Type 1 / Q000819:agonists* / Q000037:antagonists & inhibitors
D020662:Guanine Nucleotide Exchange Factors; C486707:Rapgef4 protein, mouse; D044102:Receptor, Melanocortin, Type 1; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP