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

The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian G α 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 A 2B receptor (hA 2B R) as a paradigm, a typical class A GPCR that shows promiscuous behavior in G protein coupling in this yeast system. The wild-type hA 2B R and five mutant receptors were expressed in 8 yeast strains with different humanized G proteins, covering the four major classes: G αi , G αs , G αq, and G α12 . Our experiments showed that a tyrosine residue (Y) at the C-terminus of the G α subunit plays an important role in controlling the activation of GPCRs. Receptor residues R103 3.50 and I107 3.54 are vital too in G protein-coupling and the activation of the hA 2B R, whereas L213 IL3 is more important in G protein inactivation. Substitution of S235 6.36 to alanine provided the most divergent G protein-coupling profile. Finally, L236 6.37 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.