Molecular and Cellular Pharmacology
Mutational analysis of residues important for ligand interaction with the human P2Y12 receptor

https://doi.org/10.1016/j.ejphar.2010.06.023Get rights and content

Abstract

The P2Y12 receptor, a Gi protein-coupled receptor, plays a central role in platelet activation. In this study, we did a mutational analysis of residues possibly involved in the ligand interactions with the human P2Y12 receptor. Mutant receptors were stably expressed in CHO-K1 cells with an HA-tag at the N-terminus. Expression of wild-type and mutant receptors was confirmed by detecting the HA-tag on the cell membrane. Residues in transmembrane helical domains (TMs) 3, 5, 6, and 7, which are homologous to residues important for P2Y1 receptor activation and ligand recognition, were replaced by site-directed mutagenesis. ADP-induced inhibition of forskolin-stimulated cAMP levels in the presence or absence of antagonist AR-C69931MX were investigated for each of the mutant receptors. F104S and S288P significantly increased agonist-induced receptor function without affecting the antagonism by AR-C69931MX. Arg256 in TM6 and Arg 265 in extracellular loop 3 (EL3) are more important for antagonist recognition than effect on agonist-mediated receptor function. Compared to wild-type P2Y12 receptor, mutations in Arg 256 or/and Arg 265 significantly increased the sensitivity to antagonist AR-C69931MX. Our study shows that the cytosolic side of TM3 and the exofacial side of TM5 are critical for P2Y12 receptor function, which is different from P2Y1. Arg 256 in TM6 and Arg265 in EL3 appear to play a role in antagonist recognition rather than effects on agonist-induced receptor function.

Introduction

Platelets are a fundamental component of the normal hemostatic process and abnormal platelet activation can cause thrombus formation. Upon activation, platelets change shape, aggregate and secrete granules (Jurk and Kehrel, 2005). Adenosine diphosphate (ADP), which is secreted from platelet dense granule, acts as one of the most important players to amplify the primary responses of platelets and form a stable thrombus together with generated thrombin (De Clerck and Janssen, 1990, Offermanns, 2006, Packham et al., 1987).

In platelets, ADP is an important agonist that activates platelets through Gq-coupled P2Y1 and Gi-coupled P2Y12 receptors (Daniel et al., 1998, Jin et al., 1998). Co-stimulation of P2Y1 and P2Y12 is required for ADP-induced platelet aggregation and thromboxane generation (Jin and Kunapuli, 1998). P2Y12 receptors are able to enhance other agonist-induced dense granule release (Dangelmaier et al., 2001, Storey et al., 2000). The P2Y12 receptor does not contribute to platelet shape change. However, downstream signaling events of P2Y12 receptor are essential for platelet full aggregation and thromboxane generation induced by other agonists (Kim et al., 2004, Kim et al., 2006, Shankar et al., 2006, Trumel et al., 1999). In addition, patients with defective P2Y12 receptor suffer from an abnormal ADP-induced adenylyl cyclase inhibition and platelet aggregation but retain a normal platelet shape change response (Cattaneo et al., 2003). Because of the critical role of P2Y12 in platelet activation, the thienopyridine compounds, such as clopidogrel, which target platelet P2Y12 receptor, were generated and widely used as antithrombotic drugs and have shown better benefits than aspirin in the prevention and treatment of thrombotic events (Yoneda et al., 2004).

P2Y12 is one of eight distinct functional P2Y receptors that are expressed in human tissues (Abbracchio et al., 2006). Among these P2Y receptors, P2Y1 and P2Y2 have been studied using mutagenesis and results showed that positively charged residues near the exofacial side of TM3, TM7 and TM6 of P2Y1 receptor were important for recognition of agonist and positively charged residues of TM6 and TM7 were important for agonist binding to P2Y2 receptor (Erb et al., 1995, Jiang et al., 1997). In addition, charged amino acids in EL2 (Glu209) and EL3 (Arg287) are also important for P2Y1 receptor activation (Hoffmann et al., 1999). P2Y12 and P2Y1 receptors have identical agonists: ADP and 2-methylthio-ADP (2-MeSADP), but they only have about 25% identity of amino acids in human sequences (Takasaki et al., 2001). The differences among P2Y receptors may account for differences in their ability to be recognized and activated by agonists.

In the current study, we characterized the sites for the ligand recognition and receptor activation by site-directed mutagenesis in TM3, TM5, TM6, TM7 and EL3 of the P2Y12 receptor. Inhibition of cAMP level by ADP was used as an indicator of receptor function. 5'-adenylic acid, N-[2-(methylthio) ethyl]-2-[(3,3,3-trifluoropropyl) thio]-, monoanhydride with (dichloromethylene) bis [phosphonic acid] (AR-C69931MX) was used to test the ability of mutant receptors to recognize the antagonist. The goal of this work is to provide information which may be useful in designing more selective ligands based on structural differences between the receptors.

Section snippets

Materials

FITC-labeled monoclonal antibody (HA.11) against the hemagglutinin epitope (HA-tag) was purchased from Covance Research Products (Berkeley, CA). ADP, ATP, forskolin and cAMP, were purchased from Sigma-Aldrich (St. Louis, MO). 3-isobutyl-1-methylxanthine (IBMX) was purchased from Biomol (Plymouth Meeting, PA). [3H]Adenine was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). All other reagents were reagent-grade, and deionized water was used throughout. Lipofectamine™ 2000

Construction and stable expression of wild-type and mutant human P2Y12 receptors in CHO-K1 cells

Different mutant constructs of human P2Y12 receptor were prepared as described in MATERIALS AND METHODS and the residues of human P2Y12 receptor selected for site-directed mutagenesis are shown in Fig. 1. CHO-K1 cells were transfected with the P2Y12 wild-type receptor and mutants, and clones growing from single cells which were resistant to 500 μg/ml hygromycin B, were selected. We screened the clones by detecting membrane HA-tag expression using a FITC-labeled monoclonal antibody against HA-tag

Discussion

In the present study, we investigated residues within the TMs and EL3 of human P2Y12 receptor that may be critical for agonist binding and receptor activation. Since P2Y1 and P2Y12 share the same agonist and both receptors are important for platelet activation, we generated site-direct mutant P2Y12 receptors by modifying certain amino acids, which have been shown critical for receptor function based on studies of the P2Y1 receptor (Costanzi et al., 2004, Jiang et al., 1997).

Based on the docking

Acknowledgements

This work is supported by Grants HL80444 and HL60683 from National Institutes of Health.

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