BACMAM SYSTEM FOR FRET BASED cAMP SENSOR EXPRESSION IN STUDIES OF G-PROTEIN COUPLED RECEPTORS

Olga Mazina1,2, Reet Reinart-Okugbeni1,2, Sergei Kopanchuk1,2, Ago Rinken1,2. 1University of Tartu, Institute of Chemistry, Ravila 14a, 50411, Tartu, Estonia, 2Competence Centre on Reproductive Medicine & Biology, Tiigi 61b, 50410, Tartu, Estonia

Cyclic adenosine monophosphate (cAMP) is a second messenger of many G-protein coupled receptors (GPCRs) and is thus a useful readout molecule to estimate the biological activity of various GPCR-specific agents.

Here we report the development and use of baculovirus-based BacMam transduction system for expression of a FRET biosensor for cAMP (Epac2-camps) [1,2]. The new viral transduction system is an easy and robust tool for ligand screening at second messenger level in a variety of mammalian cell lines, whereas the level of protein expression is adjustable in a dose-dependent manner depending on the viral multiplicity of infection of cells.

The functional assays were performed on B16F10 murine melanoma cell line endogenously expressing melanocortin-1 receptor (MC1R). The activation profile of the receptor was characterized by a set of full and partial agonists of MC1R.

The bivalent ions Ca2+ as well as Mg2+ modulated potencies of ligands, this effect was ligand and ion-specific.

Table: The effect of bivalent cations on MC1R activation by its agonists.

Cells were transduced with BacMam-Epac2-camps virus for 3 h and further incubated for 21 h in complete growth medium supplemented with 10 mM sodium butyrate. Cells were washed with 1 mM EDTA before the experiment. Chelating agent weas removed and cells were assayed in DPBS (with or without 1 mM Ca2+ or Mg2+) upon 10 min treatment with MC1R ligand. Responses were measured using Epac2-camp sensor FRET change. pEC50 ± standard error values are calculated from a selected representative experiment measured in duplicates with comparable results obtained from two independent replicate experiments. N.D.: not detectable.

Our results obtained for MC1R indicate that BacMam-Epac2-camps system may also be applicable for characterization of activation of other GPCRs and can be implemented for routine analysis and high throughput screening (Z´ > 0,6).

The work was funded by Estonian Ministry of Education and Science (SF0180032s12) and by the European Union through the European Regional Development Fund (TK114, 30020).

1. Mazina, O., Reinart-Okugbeni, R., Kopanchuk, S. & Rinken, A. (2012) J. Biomol. Screening (in press)

2. Nikolaev, V.O., Bünemann, M., Hein, L., Hannawacker, A. & Lohse, M.J. (2004). The J.Biol. Chem. 279, 37215-8.