A yeast screening method to decipher the interaction between the adenosine A2B receptor and the C-terminus of different G protein α-subunits.
Source: Purinergic Signal., Volume 10, Issue 3, pp. 441-53 (2014)
- Liu, R.; Groenewoud, N.J.A.; Peeters, M.C.; Lenselink, E.B.; IJzerman, A.P.
- 26 January 2014
- Online publication
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.