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Article

Functional Expression of Adenosine A3 Receptor in Yeast Utilizing a Chimera with the A2AR C-Terminus

1
Department of Chemical and Biomolecular Engineering, Tulane University, 6823 St Charles Ave, New Orleans, LA 70118, USA
2
Department of Chemical Engineering, Carnegie Mellon University, 5000 Forbes Ave, Pittsburgh, PA 15213, USA
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(12), 4547; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21124547
Received: 7 June 2020 / Revised: 21 June 2020 / Accepted: 24 June 2020 / Published: 26 June 2020
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. View Full-Text
Keywords: adenosine A3R; GPCR trafficking; yeast; GPCR signaling adenosine A3R; GPCR trafficking; yeast; GPCR signaling
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MDPI and ACS Style

Jain, A.R.; Robinson, A.S. Functional Expression of Adenosine A3 Receptor in Yeast Utilizing a Chimera with the A2AR C-Terminus. Int. J. Mol. Sci. 2020, 21, 4547. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21124547

AMA Style

Jain AR, Robinson AS. Functional Expression of Adenosine A3 Receptor in Yeast Utilizing a Chimera with the A2AR C-Terminus. International Journal of Molecular Sciences. 2020; 21(12):4547. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21124547

Chicago/Turabian Style

Jain, Abhinav R., and Anne S. Robinson 2020. "Functional Expression of Adenosine A3 Receptor in Yeast Utilizing a Chimera with the A2AR C-Terminus" International Journal of Molecular Sciences 21, no. 12: 4547. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21124547

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