Abstract
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/A2AR comprising the N-terminus and transmembrane domains from A3R (residues 1–284) and the cytoplasmic C-terminus of the A2AR (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 Gai-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.
Highlights
The adenosine A3 receptor (A3R) was the last of the four subtypes to be discovered and was the only subtype that was deorphanized after clone identification [1]
Recombinant expression of the human A3R in yeast has previously resulted in protein that is incapable of binding to its ligand or producing downstream activation [24,25]
For example, the C-terminus of A2A receptor (A2AR) contains two D/E-X-D/E motifs that would facilitate interaction with the COPII endoplasmic reticulum exit machinery [29], which is absent in A3R (Figure S1)
Summary
The adenosine A3R was the last of the four subtypes to be discovered and was the only subtype that was deorphanized after clone identification [1]. As adenosine levels increase in these conditions/diseases, therapeutic intervention targeting A3R offers promising treatment potential [5]. Because of the “two-fold nature of A3R signaling”, understanding whether activation of the receptor provided protective or harmful effect was a major challenge for therapeutic development [1]. Clinical trials for treatment of rheumatoid arthritis, plaque psoriasis, non-alcoholic steatohepatitis and hepatocellular carcinoma via agonist targeting of the A3 receptors is underway [6,7,8,9,10]. Structure-guided drug discovery will further aid in understanding of the receptor and developing highly selective drugs that minimize adverse events [6,11]
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