Functional Expression of Adenosine A₃ Receptor in Yeast Utilizing a Chimera with the A_2AR C-Terminus

The adenosine A₃ receptor (A₃R) is the only adenosine receptor subtype to be overexpressed in inflammatory and cancer cells and therefore is considered a novel and promising therapeutic target for inflammatory diseases and cancer. Heterologous expression of A₃R at levels to allow biophysical characterization is a major bottleneck in structure-guided drug discovery efforts. Here, we apply protein engineering using chimeric receptors to improve expression and activity in yeast. Previously we had reported improved expression and trafficking of the chimeric A₁R variant using a similar approach. In this report, we constructed chimeric A₃/A_2AR comprising the N-terminus and transmembrane domains from A₃R (residues 1-284) and the cytoplasmic C-terminus of the A_2AR (residues 291-412). The chimeric receptor showed approximately 2-fold improved expression with a 2-fold decreased unfolded protein response when compared to wild type A₃R. Moreover, by varying culture conditions such as initial cell density and induction temperature a further 1.7-fold increase in total receptor yields was obtained. We observed native-like coupling of the chimeric receptor to G_ai-Gpa1 in engineered yeast strains, activating the downstream, modified MAPK pathway. This strategy of utilizing chimeric receptor variants in yeast thus provides an exciting opportunity to improve expression and activity of "difficult-to-express" receptors, expanding the opportunity for utilizing yeast in drug discovery.