A yeast screening method to decipher the interaction between the adenosine A(2B) receptor and the C-terminus of different G protein alpha-subunits

作者：Liu Rongfang; Groenewoud Nick J A; Peeters Miriam C; Lenselink Eelke B; IJzerman Ad P^*

来源：Purinergic Signalling, 2014, 10(3): 441-453.

DOI：10.1007/s11302-014-9407-6

• G protein-coupled receptor
• G protein coupling
• Yeast screening
• Adenosine A(2B) receptor
• DRY motif

The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian G(alpha) 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 alpha-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(alpha i), G(alpha s), G(alpha q,) and G(alpha 12). Our experiments showed that a tyrosine residue (Y) at the C-terminus of the G(alpha) 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.

• 出版日期2014-9