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. 1997 Aug 1;40(16):2588-95.
doi: 10.1021/jm970084v.

Mutagenesis reveals structure-activity parallels between human A2A adenosine receptors and biogenic amine G protein-coupled receptors

Q Jiang  1 , B X Lee, M Glashofer, A M van Rhee, K A Jacobson
Affiliations

Mutagenesis reveals structure-activity parallels between human A2A adenosine receptors and biogenic amine G protein-coupled receptors

Q Jiang et al. J Med Chem. .

Abstract

Structure-affinity relationships for ligand binding at the human A2A adenosine receptor have been probed using site-directed mutagenesis in the transmembrane helical domains (TMs). The mutant receptors were expressed in COS-7 cells and characterized by binding of the radioligands [3H]CGS21680, [3H]NECA, and [3H]XAC. Three residues, at positions essential for ligand binding in other G protein-coupled receptors, were individually mutated. The residue V(3.32) in the A2A receptor that is homologous to the essential aspartate residue of TM3 in the biogenic amine receptors, i.e., V84(3.32), may be substituted with L (present in the A3 receptor) but not with D (in biogenic amine receptors) or A. H250(6.52), homologous to the critical N507 of rat m3 muscarinic acetylcholine receptors, may be substituted with other aromatic residues or with N but not with A (Kim et al. J. Biol. Chem. 1995, 270, 13987-13997). H278(7.43), homologous to the covalent ligand anchor site in rhodopsin, may not be substituted with either A, K, or N. Both V84L(3.32) and H250N(6.52) mutant receptors were highly variable in their effect on ligand competition depending on the structural class of the ligand. Adenosine-5'-uronamide derivatives were more potent at the H250N(6.52) mutant receptor than at wild type receptors. Xanthines tended to be close in potency (H250N(6.52)) or less potent (V84L(3.32)) than at wild type receptors. The affinity of CGS21680 increased as the pH was lowered to 5.5 in both the wild type and H250N(6.52) mutant receptors. Thus, protonation of H250(6.52) is not involved in this pH dependence. These data are consistent with a molecular model predicting the proximity of bound agonist ligands to TM3, TM5, TM6, and TM7.

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Figures

Figure 1
Figure 1
Generalized features of GPCRs, showing residue locations in TM3, TM5, and TM7 that are important for ligand binding in adenosine receptors and in biogenic amine receptors. Glycosylation occurs on the N-terminal segment (NT) in the biogenic amine receptors and on the second extracellular loop (E2) in adenosine A2A receptors. A palmitoylation site occurs in biogenic receptors at the C-terminal segment (CT) and in the adenosine A1 and A3 receptors. A sodium-binding site occurring at a D residue (2.50) modulates agonist binding. The third intracellular loop (I3), which directly contacts G proteins, contains an α-helical region. Adenosine is shown in the central cavity of the transmembrane region, which has been opened for illustration purposes. Mutagenesis of human A2A receptors indicates that the adenine moiety is largely associated with aromatic residues in TM5, TM6, and TM7. TM3 and TM7 contain residues, mutation of which selectively affects agonists, that are proposed to coordinate the ribose moiety. [Table: see text]
Figure 2
Figure 2
Structure of the agonists and antagonists used in this study. Structure numbering is included for adenosine and xanthine derivatives.
Figure 3
Figure 3
Representative competition curves for displacement of binding of [3H]CGS21680 at wild type and mutant human A2A receptors by IB-MECA (A) and XAC (B).
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