Aim: To develop a universal high-throughput screening assay based on Galpha15/16- mediated calcium mobilization for the identification of novel modulators of Gprotein- coupled receptors (GPCR).
Methods: In the present study, CHO-K1 or HEK293 cells were co-transfected with plasmids encoding promiscuous G-protein Galpha15/16 and various receptors originally coupled to Galphas, Galphai, or Galphaq pathways. Intracellular calcium change was monitored with fluorescent dye Fluo-4.
Results: We found out for all the receptors tested, Galpha15/16 could shift the receptorso coupling to the calcium mobilization pathway, and the EC50 values of the ligands generated with this method were comparable with reported values that were obtained using traditional methods. This assay was validated and optimized with the zeta-opioid receptor, which originally coupled to Galphai and was recently found to play important roles in neurodegenerative and autoimmune diseases. A largescale screening of 48 000 compounds was performed based on this system. Several new modulators were identified and confirmed with the traditional GTPgammaS binding assay.
Conclusion: This cell-based calcium assay was proved to be robust and easy to automate, and could be used as a universal method in searching for GPCR modulators.