The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts

Abstract

P2Y12, a G protein-coupled receptor that plays a central role in platelet activation has been recently identified as the receptor targeted by the antithrombotic drug, clopidogrel. In this study, we further deciphered the mechanism of action of clopidogrel and of its active metabolite (Act-Met) on P2Y12 receptors. Using biochemical approaches, we demonstrated the existence of homooligomeric complexes of P2Y12 receptors at the surface of mammalian cells and in freshly isolated platelets. In vitro treatment with Act-Met or in vivo oral administration to rats with clopidogrel induced the breakdown of these oligomers into dimeric and monomeric entities in P2Y12 expressing HEK293 and platelets respectively. In addition, we showed the predominant association of P2Y12 oligomers to cell membrane lipid rafts and the partitioning of P2Y12 out of rafts in response to clopidogrel and Act-Met. The raft-associated P2Y12 oligomers represented the functional form of the receptor, as demonstrated by binding and signal transduction studies. Finally, using a series of receptors individually mutated at each cysteine residue and a chimeric P2Y12/P2Y13 receptor, we pointed out the involvement of cysteine 97 within the first extracellular loop of P2Y12 in the mechanism of action of Act-Met.

Fig. 1.

Disruption by Act-Met of oligomeric forms of P2Y12 expressed in HEK293 cells. HEK293 cells coexpressing HA- and cMyc-tagged P2Y12 receptors were incubated for 1 h at room temperature with the indicated concentration of Act-Met, and whole-cell extracts were prepared in octyl-glucoside buffer, as described in Materials and Methods. (A) Cell extracts were subjected to SDS-PAGE under nonreducing conditions followed by anti-HA, anti-cMyc or anti-Cterm P2Y12 immunoblotting. (B) Extracts from Act-Met-treated cells (Left) were subjected SDS/PAGE under nonreducing conditions, whereas extracts from untreated cells were subjected to SDS/PAGE under reducing conditions (Right, increasing concentrations of DTT). Anti-P2Y12 immunoblotting was then performed.

Fig. 2.

Localization of functional P2Y12 oligomeric complexes in rafts and redistribution of the receptor as dimers and monomers outside membrane microdomains on treatment of P2Y12 expressing HEK293 cells with Act-Met. (A) HEK293T cells coexpressing cMyc-P2Y12 and HA-P2Y13 were incubated for 1 h at 37°C in the presence of 4 μM Act-Met or vehicle. Then, cells were lysed in 0.5% Triton X-100 containing buffer. Lysates were fractionated on a sucrose step gradient (5–40%). The centrifuged gradient was divided into eight equal fractions, and protein concentrations were determined for each fraction. Aliquots containing 2 μg of protein (fraction 2), 4 μg of protein (fraction 3), and 20 μg of protein (fractions 4–8) were alkylated with 10 mM iodoacetamide and subjected to SDS/PAGE under nonreducing conditions followed by anti-P2Y12, anti-HA or anti-caveolin immunoblotting. Gradient fraction 1 was not analyzed because it did not contain any detectable protein. (B) HEK293T cells expressing cMyc-P2Y12 were incubated for 1 h at 37°C in the presence of 4 μM Act-Met or vehicle. Cell lysate density fractionation was performed as in A. To quantify P2Y12 amount in each gradient fraction, 4-μg aliquots of protein (fractions 3–7) were subjected to SDS/PAGE under reducing conditions followed by anti-P2Y12 immunoblotting. P2Y12 monomer band intensity was measured by using a GS-800 densitometer (Kodak). Anti-caveolin immunoblotting was performed in parallel to monitor the raft-enriched fractions. Binding of [³³P]2MeS-ADP was measured on 1 μg of protein as described in Materials and Methods. Bar graphs represent the specific binding reported to the protein amount.

Fig. 3.

Expression at the cell surface, oligomerization, and sensitivity to Act-Met of P2Y12 receptors mutated at Cys 97 or Cys 175, as compared to wild-type receptors. (A) The human P2Y12 amino acid sequence contains 10 cysteine residues, four extracellular cysteine at position 17, 97, 175, and 270 (black filled circles), five cysteine residues at position 194, 208, 248, 292, and 302 within the transmembrane domains, and one intracellular cysteine residue at position 315 (gray filled circles). (B) HEK293 expressing cMyc-tagged P2Y12 receptors, either wild type (WT) or mutated at Cys 97 (C97A) or Cys 175 (C175A), were incubated for 1 h at 37°C with 4 μM Act-Met. Cells were directly lysed in octyl-glucoside containing buffer.