title: Scanning mutagenesis in a yeast system delineates the role of the NPxxY(x)(5,6)F motif and helix 8 of the adenosine A(2B) receptor in G protein coupling.
abstract: The adenosine receptor subfamily includes four subtypes: the A1, A2A, A2B and A3 receptors, which all belong to the superfamily of G protein-coupled receptors (GPCRs). The adenosine A2B receptor is the least investigated of the adenosine receptors, and the molecular mechanisms of its activation have hardly been explored. We used a single-GPCR-one-G protein yeast screening method in combination with mutagenesis studies, molecular modeling and bio-informatics to investigate the importance of the different amino acid residues of the NPxxY(x)6F motif and helix 8 in the human adenosine A2B receptor (hA2BR) activation. A scanning mutagenesis protocol was employed, yielding 11 single mutations and one double mutation of the NPxxY(x)6F motif and 16 single mutations of helix 8. The amino acid residues P287(7.50), Y290(7.53), R293(7.56) and I304(8.57) were found to be essential, since mutation of these amino acid residues to alanine led to a complete loss of function. Western blot analysis showed that mutant receptor R293(7.56)A was not expressed, whereas the other proteins were. Amino acid residues that are also important in receptor activation are: N286(7.49), V289(7.52), Y292(7.55), N294(8.47), F297(8.50), R298(8.51), H302(8.55) and R307(8.60). The mutation Y290(7.53)F lost 50% of efficacy, while F297(8.50)A behaved similar to wild type receptor. The double mutation, Y290(7.53)F/F297(8.50)Y, lost around 70% of efficacy and displayed a lower potency for the reference agonist 5'-(N-ethylcarboxamido)adenosine (NECA). This study provides new insight into the molecular interplay and impact of TM7 and helix 8 for hA2B receptor activation, which may be extrapolated to other adenosine receptors and possibly to other GPCRs.
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Rongfang
authors.initials: R
authors.lastname: Liu
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Dennis
authors.initials: D
authors.lastname: Nahon
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Beau
authors.initials: B
authors.lastname: le Roy
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Eelke B
authors.initials: EB
authors.lastname: Lenselink
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Electronic address: ijzerman@lacdr.leidenuniv.nl.
authors.forename: Adriaan P
authors.initials: AP
authors.lastname: IJzerman
mesh_terms: D058917:Adenosine A2 Receptor Antagonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D019295:Computational Biology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D043704:Receptor, Adenosine A2B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D014970:Xanthines / Q000494:pharmacology
chemical_list: D058917:Adenosine A2 Receptor Antagonists; C000592275:PSB603; D043704:Receptor, Adenosine A2B; D013449:Sulfonamides; D014970:Xanthines; D019204:GTP-Binding Proteins
keywords: Adenosine A(2B) receptor; G protein coupling; G protein-coupled receptor; Helix 8; NPxxY(x)(5,6)F motif; Yeast screening