Diastereoselectivity of the P2Y11 nucleotide receptor: mutational analysis.

background and purposeThe P2Y(11) receptor, a member of the group of metabotropic nucleotide receptors, shows a stereospecific ligand recognition of P(alpha)-substituted ATP derivatives (ATP-alpha-S isomers). These compounds are suitable candidates for the development of selective P2Y(11) receptor agonists that might be used as immune modulators. We have analysed the binding mode of ATP at the P2Y(11) receptor by molecular modeling and site-directed mutagenesis. Based on our recent findings, we decided to decipher the molecular determinants of stereoselective recognition at the P2Y(11) receptor. EXPERIMENTAL APPROACH: Two amino acid residues [Glu186 in the extracellular loop 2 and Arg268 in the transmembrane domain 6 (TM6)], which are part of the nucleotide-binding pocket, were selected and studied by mutational analyses. We expected these residues to be involved in determining the stereospecificity of the P2Y(11) receptor. KEY

resultsAfter mutation of Arg268 to alanine or glutamine, the stereospecific recognition of the ATP-alpha-S isomers at the P2Y(11) receptor was lost. In contrast, at the Glu186Ala receptor mutant, the stereoselective differentiation between these isomers was increased. On the Arg268Gln/Glu186Ala double mutant we observed no further effect, except for additivity in the decrease in potency of both isomers, as compared with the single-point mutants. CONCLUSIONS AND IMPLICATIONS: Our results show that the stereospecificity of the P2Y(11) receptor for P(alpha)-substituted ATP derivatives is largely determined by the basic residue Arg268 in TM6. This will allow the design of receptor-subtype selective ligands.