Development of a Sensitive and HTS-Compatible Reporter Gene Assay for Functional Analysis of Human Adenosine A2a Receptors in CHO-K1 Cells

Adenosine A2a receptor, a member of the G protein-coupled receptor superfamily, has been demonstrated to be an important pharmacological target. It couples to stimulatory G protein and activates adenylate cyclase upon agonist stimulation. Here we attempted to stably transfect Chinese hamster ovary (CHO-K1) cells, which lack any known subtypes of adenosine receptors, with recombinant human adenosine A2a receptors (hA2aR). Rapid down-regulation of hA2aR in a clonal cell line, CHOA2a-2, was observed over a short period of time in culture. This is consistent with other groups' findings of low expression and poor G protein coupling of this receptor in several cell systems. To facilitate pharmacological profiling for hA2aR ligand, we introduced a cyclic AMP response element (CRE)-linked β-galactosidase reporter gene into CHOA2a-2 cells to generate a stable cell line, CHOA2a-2CREβgal#26. Robust cyclic AMP signal amplification was obtained using a colorimetric assay measuring β-galactosidase activity. The EC50 of...