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 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 R103(3.50) and I107(3.54) are vital too in G protein-coupling and the activation of the hA2BR, 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.
Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, Netherlands.
Rongfang
R
Liu
Nick J A
NJ
Groenewoud
Miriam C
MC
Peeters
Eelke B
EB
Lenselink
Ad P
AP
IJzerman
D000595:Amino Acid Sequence; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D043704:Receptor, Adenosine A2B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D044385:GTP-Binding Protein alpha Subunits; D043704:Receptor, Adenosine A2B