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Purinergic Signalling

, Volume 10, Issue 3, pp 441–453 | Cite as

A yeast screening method to decipher the interaction between the adenosine A2B receptor and the C-terminus of different G protein α-subunits

  • Rongfang Liu
  • Nick J. A. Groenewoud
  • Miriam C. Peeters
  • Eelke B. Lenselink
  • Ad P. IJzermanEmail author
Original Article

Abstract

The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian Gα subunits provides a useful tool for the study of GPCR activation. In this study, we used a one-GPCR-one-G protein yeast screening method in combination with molecular modeling and mutagenesis studies to decipher the interaction between GPCRs and the C-terminus of different α-subunits of G proteins. We chose the human adenosine A2B receptor (hA2BR) as a paradigm, a typical class A GPCR that shows promiscuous behavior in G protein coupling in this yeast system. The wild-type hA2BR and five mutant receptors were expressed in 8 yeast strains with different humanized G proteins, covering the four major classes: Gαi, Gαs, Gαq, and Gα12. Our experiments showed that a tyrosine residue (Y) at the C-terminus of the Gα subunit plays an important role in controlling the activation of GPCRs. Receptor residues R1033.50 and I1073.54 are vital too in G protein-coupling and the activation of the hA2BR, whereas L213IL3 is more important in G protein inactivation. Substitution of S2356.36 to alanine provided the most divergent G protein-coupling profile. Finally, L2366.37 substitution decreased receptor activation in all G protein pathways, although to a different extent. In conclusion, our findings shed light on the selectivity of receptor/G protein coupling, which may help in further understanding GPCR signaling.

Keywords

G protein-coupled receptor G protein coupling Yeast screening Adenosine A2B receptor DRY motif 

Notes

Acknowledgement

Rongfang Liu thanks the China Scholarship Council (CSC) for her PhD scholarship. NWO provided a TOP grant to A.P. IJ. (714.011.001). The authors are grateful to Prof C.E. Müeller of Bonn University for the generous gift of [3H]PSB-603 and to Dr S. Dowell from GSK for providing the yeast strains and experimental protocols.

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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Rongfang Liu
    • 1
  • Nick J. A. Groenewoud
    • 1
  • Miriam C. Peeters
    • 1
  • Eelke B. Lenselink
    • 1
  • Ad P. IJzerman
    • 1
    Email author
  1. 1.Division of Medicinal Chemistry, Leiden Academic Centre for Drug ResearchLeiden UniversityLeidenNetherlands

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