Diastereoselectivity of the P2Y₁₁ nucleotide receptor: mutational analysis

Abstract

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

A critical look at the function of the P2Y11 receptor

“…C-terminal eGFP-tagged human and canine P2Y 11 receptors both showed similar signalling properties to the respective non-tagged receptors [6,51]. Nearly all vectors expressing human P2Y 11 described in published studies were created from the initial P2RY11 sequence arising from the fusion transcript (AF030335) [2,34,45,47,48,52,54,56,58,59,[62][63][64][65][66][67]. The sequence difference results in a slightly altered N-terminal of the P2Y 11 protein from its rightful MAANVSGAK to MDRGAK that originates from the transgenic splicing with PPAN.…”

G protein-coupled adenosine (P1) and P2Y receptors: ligand design and receptor interactions

“…Mostly, the modeling of P2YRs has focused on identifying the putative binding site and the analysis of the residues involved in the ligand binding and receptor specificity, with the aim to gain information on the ligand recognition mechanism. Site-directed mutagenesis and structure-activity relationship (SAR) analysis have been used to support and guide the modeling of the P2YRs [29][30][31][32][33][34][35][36], which has been used to identify new key residues important for the ligand binding and receptor activation [25,[37][38][39]. Several models based on different structural templates have been published for many of the P2YRs.…”

“…Nevertheless, there are now nucleotide agonists selective for P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 14 Rs and nucleotide antagonists selective for P2Y 1 and P2Y 12 Rs. The diastereoselectivity of binding of the phosphate groups of nucleotide agonists selective at the P2Y 1 , P2Y 2 , P2Y 4 and P2Y 11 Rs has been characterized [29,41,42]. Also, subtype-selective non-nucleotide antagonists have been introduced for P2Y 1 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 Rs.…”

Identification and analysis of functionally important amino acids in human purinergic 12 receptor using a Saccharomyces cerevisiae expression system

“…Although P2Y12 is a major drug target, little is known about its functional domains. Few publications [15,16] have investigated P2Y12 although many functional studies have been carried out on other P2Y receptors [17–20]. The most studied is probably P2Y1, which is closely related to P2Y12 and binds the same natural agonist, ADP [5,21–23].…”