Human P2Y₁ Receptor:  Molecular Modeling and Site-Directed Mutagenesis as Tools To Identify Agonist and Antagonist Recognition Sites

Abstract

The molecular basis for recognition by human P2Y1 receptors of the novel, competitive antagonist 2'-deoxy-N6-methyladenosine 3', 5'-bisphosphate (MRS 2179) was probed using site-directed mutagenesis and molecular modeling. The potency of this antagonist was measured in mutant receptors in which key residues in the transmembrane helical domains (TMs) 3, 5, 6, and 7 were replaced by Ala or other amino acids. The capacity of MRS 2179 to block stimulation of phospholipase C promoted by 2-methylthioadenosine 5'-diphosphate (2-MeSADP) was lost in P2Y1 receptors having F226A, K280A, or Q307A mutations, indicating that these residues are critical for the binding of the antagonist molecule. Mutation of the residues His132, Thr222, and Tyr136 had an intermediate effect on the capacity of MRS 2179 to block the P2Y1 receptor. These positions therefore appear to have a modulatory role in recognition of this antagonist. F131A, H277A, T221A, R310K, or S317A mutant receptors exhibited an apparent affinity for MRS 2179 that was similar to that observed with the wild-type receptor. Thus, Phe131, Thr221, His277, and Ser317 are not essential for antagonist recognition. A computer-generated model of the human P2Y1 receptor was built and analyzed to help interpret these results. The model was derived through primary sequence comparison, secondary structure prediction, and three-dimensional homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. We have introduced a "cross-docking" procedure to obtain energetically refined 3D structures of the ligand-receptor complexes. Cross-docking simulates the reorganization of the native receptor structure induced by a ligand. A putative nucleotide binding site was localized and used to predict which residues are likely to be in proximity to agonists and antagonists. According to our model TM6 and TM7 are close to the adenine ring, TM3 and TM6 are close to the ribose moiety, and TM3, TM6, and TM7 are near the triphosphate chain.

Structurally related nucleotides as selective agonists and antagonists at P2Y1 receptors

“…Molecular modeling of both P1 and P2Y receptors (using a rhodopsin template) and their docked ligands, based on mutagenesis results, have been carried out to interpret the findings and to suggest new ligands [12,13]. With simplified pharmacophores we are currently exploring the steric and electronic constraints of the receptor binding site, and the structural basis of receptor activation.…”

“…The features of the putative binding site identified within the TM region were consistent with both mutagenesis results and known ligand specificities. To obtain an energetically refined 3D structure of the complex, we introduced a new approach, a 'cross docking' procedure [13], which simulated the reorganization of the native receptor induced by the ligand. In order to ascertain which residues of the human P2Y 1 receptor are involved in ligand recognition, we mutated individual residues of both the TMs (3, 5, 6, and 7) and ELs 2 and 3.…”

In search of selective P2 receptor ligands: interaction of dihydropyridine derivatives at recombinant rat P2X2 receptors

“…through the incorporation of a 5-phosphonate group. A phosphonate group might act similarly to the phosphate groups of nucleotide ligands, which form putative electrostatic bonds with positively-charged groups on the P2 receptors (North and Barnard, 1997;Moro et al, 1998). The incorporation of a 5-phosphonate in the 4-phenyl-1,4-DHPs MRS 2154 and MRS 2155 (differing only in the substitution at the 2-position with methyl or phenyl) resulted not in pure antagonists, but in potentiators of the action of ATP at P2X 2 receptors.…”

“…For P1 receptors, the appropriate manipulation of the substituent groups on a 1,4-DHP ring provided antagonists of high affinity and selectivity for A 3 adenosine receptors (Jiang et al, 1997). In the present study, we have begun to extend this approach to P2 receptors, with the expectation that common elements of recognition exist at purine binding sites, whether they be for purine nucleosides (P1) or nucleotides (P2) (Moro et al, 1998). Furthermore, the fact that 1,4-DHPs are already known to block an ion channel, albeit of different structure, increases the expectation that novel interactions may occur at P2 receptor gated ion channels.…”

“…Synthetic ligands which display high potency and/or selectivity at various subtypes of P2 receptors are currently being developed through library screening and rational design approaches (Williams and Bhagwat, 1996;Fischer, 1999). The cloning of the P2X and P2Y receptors has permitted the use of biophysical and computational methods (North and Barnard, 1997;Moro et al, 1998) to characterize the drug-receptor interactions, thus aiding the drug design process.…”