Diastereoselectivity of the P2Y ₁₁ nucleotide receptor: Mutational analysis

Background and purpose: The P2Y ₁₁ receptor, a member of the group of metabotropic nucleotide receptors, shows a stereospecific ligand recognition of P _α-substituted ATP derivatives (ATP-α-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 results: After mutation of Arg268 to alanine or glutamine, the stereospecific recognition of the ATP-α-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 _α- substituted ATP derivatives is largely determined by the basic residue Arg268 in TM6. This will allow the design of receptor-subtype selective ligands.