Functional Expression of Adenosine A(3) Receptor in Yeast Utilizing a Chimera with the A(2A)R C-Terminus

The adenosine A(3) receptor (A(3)R) 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 A(3)R 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 A(1)R variant using a similar approach. In this report, we constructed chimeric A(3)/A(2A)R comprising the N-terminus and transmembrane domains from A(3)R (residues 1–284) and the cytoplasmic C-terminus of the A(2A)R (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 A(3)R. 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.