interaction with both extracellular cysteine residues ...

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Prepublished online January 30, 2003; doi:10.1182/blood-2002-10-3027

Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues, C17 and C270 Zhongren Ding, Soochong Kim, Robert T Dorsam, Jianguo Jin and Satya P Kunapuli

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Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues, C17 and C270

#§

#

§

#§

Zhongren Ding , Soochong Kim , Robert T. Dorsam* , Jianguo Jin , and Satya P. # §

Kunapuli * #

§

Department of Physiology, *Department of Pharmacology, and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA

*This work was supported by Research Grants HL60683 and HL64943 from the National Institutes of Health (S. P. K.). R.T.D. is supported by a training grant T32 HL07777 from the National Institutes of Health.

Address correspondence to: Satya P. Kunapuli, Ph.D. Department of Physiology Temple University Department of Physiology- Rm. 224 OMS 3420 N. Broad Street Philadelphia, Pennsylvania 19140 USA Phone: (215) 707-4615 Fax: (215) 707-4003 E-mail: [email protected]

Copyright (c) 2003 American Society of Hematology


ABSTRACT Human platelets express two G protein-coupled nucleotide receptors: the P2Y1 receptor, coupled to Gq, and the P2Y12 receptor, coupled to Gi. Although these two receptors have similar pharmacological profiles, they have different reactivities towards thiol reagents. The thiol agent p-chloromercuribenzenesulfonic acid (pCMBS), and the active metabolites from antiplatelet drugs, clopidogrel and CS747, inactivate the P2Y12 receptor and are predicted to interact with the extracellular cysteine residues on the P2Y12 receptor. In this study we identified the reactive cysteine residues on the human P2Y12 receptor by site-directed mutagenesis using pCMBS as the thiol reagent. C97S and C175S mutants of the P2Y12 receptor did not express when transfected into CHO-K1 cells, indicating the essential nature of a disulfide bridge between these residues. The C17S, C270S, and C17S/C270S double mutants had similar EC50 values for ADP and 2MeSADP when compared with the wild type P2Y12. Similarly, IC50 values for BzATP, an antagonist of the P2Y12 receptor, also did not differ dramatically among these mutants and the wild type P2Y12 receptor. pCMBS inactivated the wild type P2Y12 receptor in a concentration-dependent manner, whereas it had no effect on the P2Y1 receptor. Finally, pCMBS partially affected the Gi coupling of C17S or C270S receptor mutants, but had no effect on C17S/C270S P2Y12 receptor-mediated inhibition of adenylyl cyclase. These results indicate that, unlike the P2Y1 receptor, which has two essential disulfide bridges linking its extracellular domains, the P2Y12 receptor has two free cysteines in its

2


extracellular domains (C17 and C270), both of which are targets of thiol reagents. We speculate that the active metabolites of clopidogrel and CS747 form disulfide bridges with both C17 and C270 in the P2Y12 receptor, and thereby inactivate the receptor.

3


Extracellular nucleotides act on cell surface receptors, known as P2 receptors, leading to a number of physiological responses including cardiac muscle contraction and platelet aggregation 1. ATP and ADP are released from several sources in the body, including purinergic nerve endings, platelets, chromaffin cells, and endothelial cells 2. The P2 receptors are divided mainly into two classes: P2X ligand gated channels and P2Y receptors coupled to heterotrimeric G proteins 3. Several members of the P2X family and the P2Y family have been cloned 3-8. Of the P2Y receptor subtypes characterized to date, the majority couple to Gq and activate phospholipase C, but some couple to Gi and inhibit adenylyl cyclase. These Gi coupled P2Y receptors are found in platelets 9, C6 rat glioma cells

10,11

, and B10 microvascular endothelial

cells 12. Of the G protein coupled P2Y receptors, P2Y12 is expressed in platelets and in the brain 7,8, whereas the P2Y1 receptor is expressed ubiquitously

13

. The genes for P2Y12 and P2Y1

receptors are co-localized on chromosome 3q21-q25 14 and share 22% sequence identity 7,8. The P2Y1 and the P2Y12 receptors play an important role in the activation of platelets, and coactivation of both the receptors is essential for ADP-induced activation of platelet fibrinogen receptor

9,15,16

. In platelets, these two receptors have identical pharmacological profiles (ADP

and 2MeSADP agonists; ATP and BzATP antagonists) 17-20. An antiplatelet drug, clopidogrel, is metabolized in the liver to an active metabolite containing a free thiol group, which inactivates the P2Y12 receptor through formation of a disulfide bridge with extracellular cysteine residues 21-23

. Another antiplatelet drug, CS-747, also neutralizes the P2Y12 receptor by the same

4


mechanism 24,25. In addition, the P2Y12 receptor is irreversibly antagonized by the thiol reagent pCMBS 22,26,27. Most G protein-coupled receptors (GPCRs), including the P2Y receptors, possess two conserved extracellular cysteine residues located on the first and second extracellular loops (Fig. 1). A disulfide bridge formed between these two cysteines is essential for the function of a number of GPCRs

, including rhodopsin, the TSH-releasing hormone receptor, the

thromboxane receptor, the GnRH receptor, and the P2Y1 receptor

28-30

. In addition to this

essential disulfide bridge between C124 in the first extracellular loop and C202 in the second extracellular loop of the P2Y1 receptor, Hoffmann et al 31 found an additional essential disulfide bridge between two other extracellular cysteines (C42 in the amino terminal and C296 in the third extracellular loop) of the P2Y1 receptor. Although the P2Y12 receptor also has these corresponding cysteine residues (C17 and C270) in its extracellular regions (Fig. 1), the function of these cysteines has not been investigated. In this study, we evaluated the function of extracellular cysteine residues of the P2Y12 receptor by site-directed mutagenesis and conclude that there is no essential disulfide bridge between C17 and C270. Furthermore, we conclude that thiol reagents such as pCMBS can inactivate the P2Y12 receptor by targeting either C17 or C270 or both. Thus, we postulate that C17 and C270 are possible targets of the antithrombotic drugs, clopidogrel and CS-747, through the action of an active metabolite.

5


EXPERIMENTAL PROCEDURES:

Materials: pCMBS (p-chloromercuribenzenesulfonic acid) was purchased from Toronto Research Chemicals Inc. Anti-FLAG M2 monoclonal antibody FITC conjugate, ADP, 2methylthio-ADP (2-MeSADP), and 2’-& 3’-O-(4-benzoylbenzoyl) adenosine 5’-triphosphate (BzATP) were from Sigma Chemical Co. (St. Louis, MO). Slow Fade Light Antifade Kit was purchased from Molecular Probes (Eugene, OR). Lab-Tek Chamber Slide Culture Chambers were purchased from Nunc (Naperville, IL). pcDNA3.1/Hygro(+) vector was purchased from Invitrogen (Carlsbad, CA). Ready-to-Go PCR beads were purchased from Amersham Pharmacia Biotech Inc (Piscataway, NJ). QuickChange Site-Directed Mutagenesis Kit was 3

14

obtained from Stratagene (La Jolla, CA). Myo-2-[3H] inositol, [ H] adenine and [ C] cAMP were from NEN Life Science Products (Boston, MA).

Construction of the P2Y12 expression plasmid: Total RNA was isolated from human platelets by the RNAzol procedure (Tel-Test Inc., Friendswood, TX), and cDNA was prepared from 5 µ g of total RNA using a first strand synthesis kit (Gibco-BRL, Gaithersberg, MD). The PCR was carried out using forward and reverse primers specific for human P2Y12 receptor cDNA [GenBank Accession No: AF313449] 8. The nucleotide sequence encoding the FLAG epitope (DYKDDDDK) was inserted at the beginning of the translation initiation. The sense primer containing a Hind III restriction site and the FLAG epitope sequence is 5’GCGCAAGCTTACCATGGACTACAAAGACGATGACGACAAGCAAGCCGTCGACAATC TC-3’.

The

antisense

primer

containing 6

Xba

I

restriction

sites

is

5'-


GCTCTAGACATTGGAGTC-TCTTCATTTGG-3'.

The

restriction

enzyme

sites

are

underlined, and the coding sequence of FLAG epitope is given in bold letters. After an initial denaturation for 5 min at 94°C, the amplifications were carried out for 35 cycles using Ready to Go PCR beads (Pharmacia) as follows: denaturation at 94 °C for 45 sec, annealing at 50°C for 45 sec, and extension at 72°C for 1 min. The final cycle was followed by an additional extension for 7 min at 72°C. An expression plasmid (pcDNA3-HP2Y12) was constructed in pcDNA3.1-Hygro (+) vector by digesting the RT-PCR product with Hind III and Xba I and inserting it into the vector digested with the same set of restriction enzymes. The nucleotide sequence of the P2Y12 receptor cDNA in the expression plasmid was confirmed by DNA sequence analysis.

Site-directed mutagenesis: All mutations were introduced into pcDNA3-HP2Y12 using the QuickChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). The resulting changes in oligonucleotides and the corresponding amino acids are shown in Table 1. Using C270S as a template and C17S mutation primers, a C17S/C270 double mutation was achieved by the same method. All mutations were confirmed by DNA sequencing.

Cell culture: Chinese hamster ovary (CHO-K1) cells were grown in HAM’s F12 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin/amphotericin at 37°C with 5% CO2. CHO-K1 cells transfected with wild type or mutant P2Y12 receptors were grown in the same medium supplemented with 400 µg/ml hygromycin. Astrocytoma cells (1321N1)

7


stably expressing the human P2Y1 receptor Medium

(DMEM)

supplemented

32

with

were grown in Dulbecco's Modified Eagle's 10%

fetal

bovine

serum,

1%

penicillin/streptomycin/amphotericin, and 500µg/ml G418 at 37°C with 5% CO2. Experiments were carried out in confluent cultures 2 days after plating in 6- or 12-well plates.

Stable expression of human P2Y12 receptor in CHO-K1 cells: The expression construct for the wild type P2Y12 receptor (pcDNA3-HP2Y12) or for each of the P2Y12 site-directed mutants (C17S, C97S, C175S, C270S, and C17S/C270S) (1 µg) was used to transfect CHO-K1 cells using lipofectamine as described previously

33

. CHO-K1 cells were also transfected with

pcDNA3.1/Hygro(+) to serve as a mock control. The growth medium was replaced after 6 h with fresh medium containing 400 µg/ml hygromycin. Stable transfectants were selected on medium containing 400 µg/ml hygromycin and screened for the expression of wild type or mutant P2Y12 receptor by second messenger (cAMP) inhibition.

FLAG tag detection in CHO-K1 cells by immunofluorescence microscopy: Cells were cultured in Lab-Tek Chamber Slide Culture Chambers overnight and washed twice with TBS (Tris buffered saline: 0.05M Tris, 0.15 M NaCl, pH 7.4). Cells were fixed with freshly prepared methanol: acetone (1:1, using a glass vial) for 1 minute at room temperature, and then washed with TBS four times and incubated with anti-FLAG M2-FITC at 40 µg/ml in TBS at room temperature for 2 hours (gentle shaking in the dark). Cells were further washed twice with TBS and 3 drops of component C of Slow Fade Light Antifade Kit were added and incubated for 10 minutes at room temperature. Finally, component C was washed away with TBS, a small drop of component A was added, then covered with a cover slip and examined using a Nikon Eclipse 8


TE300 fluorescence microscope (Melville, NY).

3

Cyclic AMP assay: Cells were cultured in 6-well plates and labeled with 2 µl/ml [ H]-adenine (74 kBq/ml) for 4 h or overnight at 37°C. The radiolabeling medium was replaced by fresh growth medium containing 1 mM IBMX and incubated for 10 min at 37°C. After stimulation with 20 µM forskolin for 1 min, various concentrations of agonist and antagonist were added and incubated at 37°C for 10 min unless indicated. The reactions were terminated by addition of 14

1 M HCl containing 2000 cpm of [ C]- cyclic AMP (2 GBq/mmol) as a recovery standard. Cyclic AMP levels were determined as described earlier

32,34

and expressed as a percentage of

3

total [ H]-adenine nucleotides. The EC50/IC50 values are calculated using Kaleidagraph 3.5 fitting the curve to m1+(m2-m1)/(1 + (m0/m3)^m4), where m0 is the difference between the maximal and minimal response, m1 is the minimal value of response, m2 is the maximal value of response, m3 is the estimated EC50 or IC50, and m4 is the slope. After fitting the curve, the calculated mean and SEM of EC50 or IC50 is given automatically by the program.

Measurements of Inositol Phosphates: Inositol phosphates were measured essentially as described 35,36. Confluent cultures of cells in 12-well plates were labeled with 1 µCi/ml of myo2-[3H] inositol in inositol-free DMEM for 24 h. Labeled cells were washed once, the medium was replaced with 890 µl of fresh inositol-free 20 mM HEPES-buffered Eagle’s medium, pH 7.4, and the cells were incubated at 37°C for 30 min before proceeding; this step helps to reduce background levels of [3H] inositol phosphates, so that agonist-stimulated accumulation could be detected more easily. After incubation for 30 min at 37°C, 10 µl of 1 M LiCl were added to a final concentration of 10 mM and the incubation continued for an additional 10 min.

9


The cells were stimulated with 100 µl of ADP at a final concentration of 10 µM for 15 min and the reaction was terminated by aspiration of the medium, addition of 0.75 ml of 10 mM formic acid and incubation at room temperature for 30 min. The solution containing the extracted inositol phosphates were neutralized by dilution with 3 ml of 10 mM NH4OH (yielding a final pH of 8-9) and then applied directly to a column containing 0.7 ml of the anion exchange resin, AG 1-X8 equilibrated with 40 mM ammonium formate. The column was washed with 4 ml of 40 mM ammonium formate, pH 5.0, to remove the free inositol and the glyceroinositol. Total inositol phosphates were eluted with 4 ml of 2 M ammonium formate, pH 5.0. One ml of the eluate was removed and counted with 9 ml of scintillation fluid. Statistical Analysis: All experiments were performed at least three times in duplicate. Data are expressed as means +/- SE. Statistical significance was determined using the student’s t-test and significance was designated to p-values < 0.05.

RESULTS AND DISCUSSION Human platelets express two P2Y receptors, the P2Y1 and P2Y12 receptors,

7,8,13

and

each have four conserved extracellular cysteines capable of forming two disulfide bridges to stabilize the three dimensional structure of the receptor (Fig.1). In the case of the P2Y1 receptor, C124 in the first extracellular loop forms a disulfide bridge with C202 in the second extracellular loop which is essential for the function of the receptor

31

. In addition, the P2Y1

receptor has an additional disulfide bridge between C42 in the amino terminus and C296 in the third extracellular loop 31. Disruption of this bridge by site-directed mutagenesis of either C42

10


or C296 results in dramatic loss of the ability of the agonist to activate the receptor 31. As both the P2Y1 and the P2Y12 receptors have similar pharmacological profiles in platelets

9,14,18,20

,

we first sought to determine whether the P2Y12 receptor also has two essential disulfide bridges between its four conserved extracellular cysteine residues. Secondly, we wanted to identify the cysteines in the P2Y12 receptor that interact with thiol reagents. To achieve these objectives, we constructed an expression plasmid for the P2Y12 receptor and mutated each of the four cysteines in the P2Y12 receptor (C17, C97, C175, and C270) to serines. In addition, we generated a double mutant of C17S and C270S by site-directed mutagenesis.

Stable expression of wild type and mutant human P2Y12 receptors in CHO-K1 Cells: CHOK1 cells were transfected with the P2Y12 wild type receptor and clones resistant to 400 µg/ml hygromycin were selected. We screened our clones based on their response to 10 µM ADP in the adenylyl cyclase inhibition assay. One wild type clone was selected from 16 cell clones, designated P2Y12WT. The expression of P2Y12 wild type on the surface of CHO-K1 cells was also confirmed by immunofluorescence microscopy using a monoclonal antibody directed against the FLAG tag at the N-terminus of the receptor (Fig 2). Unfortunately, this monoclonal antibody was not useful in detecting the receptor expression by the ELISA method.

Similarly, CHO-K1 cells transfected with expression constructs for P2Y12 mutants C17S and C270S were grown in the presence of hygromycin and screened by cAMP inhibition assay. Two cell clones designated P2Y12C17S and P2Y12C270S were selected from 15 and 12

11


cell clones, respectively. In contrast to the P2Y12 wild type and mutants C17S, C270S, and C17S/C270S, no positive clone was found after screening 61 cell clones stably transfected with C97S and 49 cell clones stably transfected with C175S. Immunofluorescence experiments with the antibody against the FLAG tag failed to detect any receptor expression on these cell clones. These results were not totally unexpected as similar results were obtained when C124 and C202 in the P2Y1 receptor were mutated to serines

31

. These results indicate that a disulfide bridge

between C97 and C175 might be essential for the receptor expression as is the case with the P2Y1 receptor

31

. However, it is also possible that a cysteine is required at both of these

positions for the receptor expression. Finally, one cell clone, designated P2Y12C17S/C270S, was selected from 12 hygromycin resistant clones by a similar method from CHO-K1 cells transfected with expression constructs for the P2Y12 receptor double mutant C17S/C270S. Characterization of cloned human P2Y12 receptor and the mutants expressed in CHO-K1 cells: The mutation of C42 and C296 in the P2Y1 receptor leads to dramatically diminished responses to the agonist 31. We evaluated the function of the corresponding cysteines in the P2Y12 receptor with C17S mutant and C270S mutant and compared them to the wild type P2Y12 receptor. The wild type P2Y12 receptor, the P2Y12C17S, or the P2Y12C270S mutant caused similar inhibition of forskolin-stimulated adenylyl cyclase upon stimulation by ADP (Fig. 3A) or 2MeSADP (Fig. 3B) in a concentration-dependent manner. The EC50 values for these agonists are comparable in the wild type and mutant P2Y12 receptors (Table 2). We generated a cell line stably expressing the double mutant P2Y12C17S/C270S and evaluated its 12


ability to respond to the agonists. As shown in Fig. 3, this double mutant also has similar EC50 values compared to the wild type receptor (Table 2). These results indicate that mutations at C17 and/or C270 did not considerably alter the function or coupling of the P2Y12 receptor. Similar mutations in the P2Y1 receptor dramatically increased the EC50 values of the agonists 31

. Thus, we conclude that, unlike in the P2Y1 receptor, a disulfide bridge between the cysteines

in the amino terminus (C17) and the third extracellular loop (C270) is not essential for the function of the P2Y12 receptor.

Effect of antagonist on the wild type and mutant P2Y12 receptors: Human platelets express both the P2Y1 and P2Y12 receptors that contribute to various agonist-induced physiological responses

9,15,16,37

. BzATP inhibits both the Ca2+- and cAMP-dependent intracellular signaling

pathways of ADP in endothelial cells and platelets through antagonism of the P2Y1 and P2Y12 receptors

17

. We evaluated the effect of this non-selective antagonist on the wild type and

mutant P2Y12 receptors. As shown in Fig. 4, BzATP blocks 1 µM ADP-induced inhibition of adenylyl cyclase in CHO-K1 cells stably expressing P2Y12WT, P2Y12C17S, P2Y12C270S or P2Y12C17S/C270S in a concentration-dependent manner. The IC50 values for the antagonism by BzATP are 116 ± 23 µM (wild type), 85 ± 6 µM (P2Y12C17S), 137 ± 15 µM (P2Y12C270S), and 58 ± 5 µM (P2Y12C17S/C270S). Compared with the wild type, the concentration-response curves of C17S/C270S mutant slightly shifted to the left, suggesting that 13


double mutation renders the receptor more sensitive to the antagonist. It is possible that these two extracellular cysteines play a role in the binding of the BzATP and thereby alter the sensitivity of the receptor to the antagonist.

Effects of pCMBS on the function of the P2Y receptors: A critical role of cysteine residues in the function of the platelet ADP receptor has been suggested by the ability of pCMBS, a nonpenetrating thiol reagent, to abolish ADP responses in platelets

20,26

. It was also shown that

pCMBS affects the binding of the agonist to the ADP receptor mediating the adenylyl cyclase inhibition response

22,27

. As both the P2Y1 and P2Y12 receptors have four conserved

extracellular cysteine residues (Fig. 1), we evaluated the effect of pCMBS on these two platelet P2Y receptors. As shown in Fig. 5, pCMBS blocked ADP-induced inhibition of forskolinstimulated adenylyl cyclase in the cells expressing the human P2Y12 receptor in a concentration-dependent manner, whereas it did not inhibit agonist-induced inositol phosphate formation in the cells stably expressing the P2Y1 receptor. The effect of pCMBS on the human P2Y12 receptor is consistent with the data published by Savi et al 22. Thus, pCMBS inactivates the P2Y12 receptor but not the P2Y1 receptor. As this thiol reagent is cell-impermeable and covalently binds to the free thiol groups, we predict that the C17 and C270 in the P2Y12 receptor exist as free cysteines and may be the target of pCMBS. On the other hand, the P2Y1 receptor does not have free extracellular cysteines, as the corresponding cysteines (C42 and C296) form an essential disulfide bridge, and hence is insensitive to pCMBS. Thus, we conclude that the P2Y12 receptor does not have a disulfide bridge between C17 and C270 and

14


that these residues are free cysteines in the native receptor and may be the target of the thiol reagents. The sensitivity of the P2Y12 receptor to thiol reagents explains the mechanism of clopidogrel 22,23. Since its approval in 1988, clopidogrel has been broadly used as an antiplatelet drug

21

. Although the P2Y12 receptor was thought to be the target of clopidogrel

mechanism of action remained unclear until recently

22,23,39

38

, its

. P2Y12 receptors play an essential

role in ADP-induced platelet aggregation 40 and hence, the P2Y12 receptor-defective patients 4143

and mice deficient in the P2Y12 receptor have abnormal platelet aggregation 16. Savi et al

23

proposed that the extracellular cysteine on P2Y12 was modified by formation of a disulfide bridge with the thiol group on the active metabolite of clopidogrel. Similarly, CS747 was also predicted to target the extracellular cysteines in the P2Y12 receptor through conversion to an active metabolite in the liver 24,25.

Effects of pCMBS on the wild type and mutant P2Y12 receptors: The active metabolite of clopidogrel has to be purified into an active enantiomer metabolite from the liver microsomes and is highly unstable

22,23

. Hence, we used pCMBS as a tool to identify the reactive cysteine

residue on the P2Y12 receptor using ADP-induced inhibition of adenylyl cyclase assay on CHO-K1 cells stably expressing the wild type and mutant P2Y12 receptors. As shown in Fig 6, pCMBS (20 µM) significantly reversed the inhibitory effects of 10 µM ADP on adenylyl cyclase in CHO-K1 cells stably expressing wild type and C17S (41 ± 5 vs 83 ± 12, 37.1 ± 2.7 vs 15


74 ± 3, respectively). Although pCMBS reversed the effect of ADP in the P2Y12C270S mutant, the effect was not as dramatic (39.4 ± 2.1 vs 57.3 ± 2.3). Finally, pCMBS did not reverse the ADP-induced inhibition of adenylyl cyclase in the cells expressing the double mutant P2Y12C17S/C270S (46.2 ± 2.9 vs 51 ± 3, mean ± SE, p < 0.05). These results indicate that both C17 and C270 are the targets of pCMBS. The primary target appears to be C270 as the effect of pCMBS appears to be more dramatic on the P2Y12C17S mutant than on the P2Y12C270S mutant. However, a small effect on the single C270S mutant shows that even C17 is a target of the thiol reagent. Thus, we conclude that both C17 and C270 are the targets of the thiol reagents as they exist as the free cysteine residues in the native receptor in platelets. As the active metabolites of clopidogrel and CS747 form a disulfide bridge with the free thiol groups on the P2Y12 receptor, we postulate that C17 and C270 are the targets of the metabolites of these drugs. It should be noted that in order to achieve a complete effect, a thiol reagent should block both the cysteine residues. Thus partial inhibition, such as that seen in humans when lower doses of clopidogrel are used 21,44, could be due to incomplete blockade of the C17 and C270 on the platelet P2Y12 receptor. Our results indicate that only one disulfide bridge is formed among the four extracellular cysteine and that two free extracellular cysteines are the targets of clopidogrel. Two disulfide bridges are formed among the four extracellular cysteines on the platelet P2Y1 receptor and both of them are essential to receptor function receptor

39,45

31

. Clopidogrel specifically targets the P2Y12

. Our results successfully explain the specificity of clopidogrel on P2Y12 rather

than P2Y1. As is the case with pCMBS, we believe that lack of free cysteines in the P2Y1 16


receptor makes it insensitive to clopidogrel. As the homology and positioning of the cysteine residues in the P2Y12 receptor relative to the P2Y1 receptor suggest a disulfide bond should also exist in the P2Y12 receptor, it is possible that there may be a dynamic redox exchange operating between C17 and C270. Thus, a P2Y12 receptor specific disulfide reductase operating in the external oxidative cellular environment could generate free cysteine residues from a disulfide bridge between C17 and C270 that could then be targeted by the thiol reagents. There is considerable evidence to suggest that the metabolites of clopidogrel and CS-747 have specificity for the P2Y12 receptor. Although both P2Y1 and P2Y12 receptors have similar pharmacological profiles, only the P2Y12 receptor is affected by the metabolites of clopidogrel and CS-747. This observation indicates that the specific ligand binding pocket of the P2Y12 receptor might have only one of the cysteine residues (either C17 or C270) that would be accessible for modification by the metabolite. However, such modification of a single cysteine residue by the active metabolite of clopidogrel might be sufficient to block the binding of the agonist. It is possible that pCMBS did not achieve complete inhibition because of its small size in the single mutants, whereas clopidogrel metabolite might achieve this by interacting with only one of the cysteines because of its larger size. Hence, we would speculate that the active metabolites of clopidogrel and CS-747 might interact with only one of the cysteine residues (very likely C270) in the P2Y12 receptor to cause blockade of agonist binding.

Unlike the thiol reagent pCMBS, the active metabolites of clopidogrel or CS747, with a free thiol group, could also break disulfide bridges. Thus, these active metabolites could break

17


the essential disulfide bridge between C97 and C175 of the P2Y12 receptor, thereby forming a separate disulfide bridge with each of these residues. Such action could result in an inactive receptor as the naturally formed disulfide bridge maintains the receptor in a conformation required for agonist binding. However, we believe that this is an unlikely mechanism. If the clopidogrel metabolite can break an existing disulfide bridge, then we expect that it also should inactivate a number of G protein-coupled receptors, including the P2Y1 receptor by the same mechanism. Even if it is argued that the metabolite depends on an agonist binding pocket, we expect that the P2Y1 receptor should also be inactivated by this metabolite. However, despite having the same agonist profile, the P2Y1 receptor is insensitive to clopidogrel. In conclusion, we have shown that unlike the P2Y1 receptor, which has two essential disulfide bridges in its extracellular domains, the P2Y12 receptor has two free cysteines in its extracellular domains (C17 and C270), both of which are the targets of thiol reagents. We predict that the P2Y1 receptor is insensitive to the thiol reagents and to the active metabolites of clopidogrel and CS747, because of the lack of these free cysteines in the extracellular domains. Thus, we speculate that the active metabolites of clopidogrel and CS747 form disulfide bridges with C17 and/or C270 in the P2Y12 receptor and thereby inactivate the receptor.

Acknowledgements: We thank Ms. Mayosha Mendis for her technical assistance and Drs. James L. Daniel, Barrie Ashby, A. Koneti Rao, Lee-Yuan Liu-Chen, Fujio Sekiya, and Todd M. Quinton, Temple University Medical School, for critically reading the manuscript.

18


19


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Ayyanathan K, Naylor SL, Kunapuli SP. Structural characterization and fine

chromosomal mapping of the human P2Y1 purinergic receptor gene (P2YR1). Som. Cell Mol. Genet. 1996;22:419-424. 15. Fabre JE, Nguyen M, Latour A, Keifer JA, Audoly LP, Coffman TM, Koller BH. Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice. Nat Med. 1999;5:1199-1202

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16. Foster CJ, Prosser DM, Agans JM, Zhai Y, Smith MD, Lachowicz JE, Zhang FL, Gustafson E, Monsma FJ, Jr., Wiekowski MT, Abbondanzo SJ, Cook DN, Bayne ML, Lira SA, Chintala MS. Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs. J Clin Invest. 2001;107:1591-1598. 17. Vigne P, Hechler B, Gachet C, Breittmayer JP, Frelin C. Benzoyl ATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation. Biochem Biophys Res Commun. 1999;256:94-97 18.

Hourani SMO, Hall DA. Receptors for ADP on human platelets. Trends in

Pharmacol. Sci. 1994;15:103-108 19. Kunapuli SP. Molecular Physiology of platelet ADP receptors. Drug Development Research. 1998;45:135-139 20. Mills DCB. ADP receptor in platelets. Thromb. Haemost. 1996;76:835-856 21. Bennett JS. Novel platelet inhibitors. Annu Rev Med. 2001;52:161-184 22. Savi P, Labouret C, Delesque N, Guette F, Lupker J, Herbert JM. P2Y12, a new platelet ADP receptor, target of clopidogrel. Biochem Biophys Res Commun. 2001;283:379383. 23. Savi P, Pereillo JM, Uzabiaga MF, Combalbert J, Picard C, Maffrand JP, Pascal M, Herbert JM. Identification and biological activity of the active metabolite of clopidogrel. Thromb Haemost. 2000;84:891-896. 24. Sugidachi A, Asai F, Ogawa T, Inoue T, Koike H. The in vivo pharmacological profile of CS-747, a novel antiplatelet agent with platelet ADP receptor antagonist properties. Br J Pharmacol. 2000;129:1439-1446

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25. Sugidachi A, Asai F, Yoneda K, Iwamura R, Ogawa T, Otsuguro K, Koike H. Antiplatelet action of R-99224, an active metabolite of a novel thienopyridine-type G(i)-linked P2T antagonist, CS-747. Br J Pharmacol. 2001;132:47-54 26. Rao AK, Kowalska MA. ADP-induced platelet shape change and mobilization of cytoplasmic ionized calcium are mediated by distinct binding sites on platelets: 5'-pfluorosulfonylbenzoyladenosine is a weak platelet agonist. Blood. 1987;70:751-756 27. Cristalli G, Mills DCB. Identification of a receptor for ADP on blood platelets by photoaffinity labelling. Biochem.J. 1993;291:875-881 28.

Cook JV, Eidne KA. An intramolecular disulfide bond between conserved

extracellular cysteines in the gonadotropin-releasing hormone receptor is essential for binding and activation. Endocrinology. 1997;138:2800-2806. 29. D'Angelo DD, Eubank JJ, Davis MG, Dorn GW, 2nd. Mutagenic analysis of platelet thromboxane receptor cysteines. Roles in ligand binding and receptor-effector coupling. J Biol Chem. 1996;271:6233-6240. 30. Ji TH, Grossmann M, Ji I. G protein-coupled receptors. I. Diversity of receptorligand interactions. J Biol Chem. 1998;273:17299-17302 31. Hoffmann C, Moro S, Nicholas RA, Harden TK, Jacobson KA. The role of amino acids in extracellular loops of the human P2Y1 receptor in surface expression and activation processes. J Biol Chem. 1999;274:14639-14647. 32. Jin J, Tomlinson W, Kirk IP, Kim YB, Humphries RG, Kunapuli SP. The C6-2B glioma cell P2Y(AC) receptor is pharmacologically and molecularly identical to the platelet P2Y(12) receptor. Br J Pharmacol. 2001;133:521-528.

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33. Akbar GKM, Dasari VR, Webb TE, Ayyanathan K, Pillarisetti K, Sandhu AK, Athwal RS, Daniel JL, Ashby B, Barnard EA, Kunapuli SP. Molecular cloning of a novel P2 purinoceptor from human erythroleukemia cells. J. Biol. Chem. 1996;271:18363-18367 34. Daniel JL, Dangelmaier C, Jin J, Ashby B, Smith JB, Kunapuli SP. Molecular basis for ADP-induced platelet activation I: Evidence for three distinct ADP receptors on platelets. J. Biol. Chem. 1998;273:2024-2029 35. Kunapuli P, Lawson JA, Rokach J, Fitzgerald GA. Functional characterization of the ocular prostaglandin F2 (PGF2 ) receptor. Activation by the isoprostane, 12-iso-PGF2 . J. Biol. Chem. 1997;272:27147-27154 36. Filtz TM, Li Q, Boyer JL, Nicholas RA, Harden TK. Expression of a cloned P2y purinergic receptor that couples to phospholipase C. Mol.Pharmacol. 1994;46:8-14 37. Leon C, Hechler B, Freund M, Eckly A, Vial C, Ohlmann P, Dierich A, LeMeur M, Cazenave JP, Gachet C. Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice [see comments]. J Clin Invest. 1999;104:1731-1737 38. Mills DCB, Puri RN, Hu C-J, Minnitti C, Grana G, Freedman M, Colman RF, Colman RW. Clopidogrel inhibits the binding of ADP analogues to the receptor mediating inhibition of platelet adenylate cyclase. Atheroscler. Thromb. 1992;12:430-436 39. Geiger J, Brich J, Honig-Liedl P, Eigenthaler M, Schanzenbacher P, Herbert JM, Walter U. Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signaling by the antiplatelet drug clopidogrel. Arterioscler Thromb Vasc Biol. 1999;19:2007-2011. 40. Jin J, Kunapuli SP. Co-activation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc. Natl. Acad. Sci. U. S. A. 1998;95:80708074

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41. Cattaneo M, Lecchi A, Randi AM, McGregor JL, Mannucci PM. Identification of a new congenital defect of platelet aggregation characterized by severe impairment of platelet responses to adenosine 5'-diphosphate. Blood. 1992;80:2787-2796 42. Cattaneo M, Lecchi A, Lombardi R, Gachet C, Zighetti ML. Platelets from a patient heterozygous for the defect of P2CYC receptors for ADP have a secretion defect despite normal thromboxane A2 production and normal granule stores: further evidence that some cases of platelet 'primary secretion defect' are heterozygous for a defect of P2CYC receptors. Arterioscler Thromb Vasc Biol. 2000;20:E101-106. 43. Nurden P, Savi P, Heilmann E, Bihour C, Herbert J-M, Maffrand J-P, Nurden A. An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. J.Clin.Invest. 1995;95:1612-1622 44. Seyfarth HJ, Koksch M, Roethig G, Rother T, Neugebauer A, Klein N, Pfeiffer D. Effect of 300- and 450-mg clopidogrel loading doses on membrane and soluble P-selectin in patients undergoing coronary stent implantation. Am Heart J. 2002;143:118-123 45. Gachet C, Stierle A, Cazenave JP, Ohlmann P, Lanza F, Bouloux C, Maffrand JP. The thienopyridine PCR 4099 selectively inhibits ADP-induced platelet aggregation and fibrinogen binding without modifying the membrane glycoprotein IIb-IIIa complex in rat and in man. Biochem Pharmacol. 1990;40:229-238.

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Table 1. Primers for site directed mutagenesis of the P2Y12 receptor Amino acid and position C17

C97

C175

C270

Nucleotide change TGC to TCC TGT to TCT TGC to TCC TGC to TCC

Amino acid Sense change Cys to + Ser Cys to Ser Cys to Ser Cys to Ser

Primers used 5’-GGAACACCAGTCTGTCCACCAGAGACTAC-3’

+

5’-GTAGTCTCTGGTGGACAGACTGGTGTTCC-3’

+

5’-GACGGAGGTAACTTGAGACACAAAAGTTCTCAGTGGTCC-3’

+

5’-GATTTAAGGAAAGAGGATTTCTTCACATTCTTGTCTCTCGGC-3’ 5’-CCCGGGATGTCTTTGACTCCACTGCTGAAAATAC-3’

-

5’-GTATTTTCAGCAGTGGAGTCAAAGACATCCCGGG-3’

5’–GGACCACTGAGAACTTTTGTGTCTCAAGTTACCTCCGTC-3’

5’-GCCGAGAGACAAGAATGTGAAGAAATCCTCTTTCCTTAAATC-3’

Table 2. Agonist-stimulated adenylyl cyclase inhibition by the wild type P2Y12 and its mutants. EC50 ADP (µM) 0.32 ± 0.08

2-MeSADP (nM) 0.17 ± 0.03

P2Y12C17S

0.32 ± 0.09

0.20 ± 0.03

P2Y12C270S

0.26 ± 0.10

0.13 ± 0.02

0.26 ± 0.16 NAa

0.47 ± 0.16

P2Y12WT

P2Y12C17S/C270S P2Y12C97S P2Y12C175S

NAb

The values are mean ± SE, n = 3- 7. a NA: no activity or expression detectable among 61 clones tested. b NA: no activity or expression detectable among 49 clones tested.

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FIGURE LEGENDS Fig. 1. Alignment of the human P2Y1 and P2Y12 receptors: The alignment of the cloned human platelet P2Y receptors

7-9,13

was performed using a GCG program (GCG Software,

Wisconsin). The conserved cysteine residues in the extracellular domains are indicated by an arrow. Putative transmembrane domains are underlined.

Fig. 2. Cell surface expression of the FLAG-tagged human P2Y12 receptor on CHO-K1 cells. The CHO-K1 cells transfected with A) vector alone or B) the FLAG-tagged human P2Y12 receptor cDNA construct were treated with FITC-conjugated anti-FLAG antibodies and visualized in a fluorescence microscope after washing.

Fig. 3. Concentration-response curve of A) ADP- or B) 2MeSADP- induced inhibition of adenylyl cyclase stimulated with 20 µM forskolin in CHO-K1 cells stably expressing either P2Y12WT, P2Y12C17S, P2Y12C270S or P2Y12C17S/C270S. Data were normalized to the maximal response obtained in the absence of ADP or 2MeSADP (mean ± SE, n = 3 - 7).

Fig. 4. Effect of BzATP on the wild type and mutant P2Y12 receptors: BzATP antagonizes 1 µM ADP- induced inhibition of adenylyl cyclase in CHO-K1 cells stably expressing either P2Y12WT, P2Y12C17S, P2Y12C270S or P2Y12C17S/C270S stimulated with 20 µM forskolin. Data were normalized to the maximal response obtained with antagonist (mean ± SE, n = 3).

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Fig. 5. Effects of pCMBS on the P2Y1 and P2Y12 receptors. A) Cells stably expressing the P2Y12 receptor were pretreated with different concentrations of pCMBS for 10 min at 37°C before addition of 20 µM forskolin. Data were normalized to the value obtained with forskolin alone, taken as 100%, and expressed as mean ± SE (n = 4 - 6). B) Cells stably expressing the P2Y1 receptor were pretreated with 20 µM pCMBS for 10 min at 37°C and stimulated with 10 µM ADP and the total inositol phosphates were measured as described in the methods section. Data are expressed as % of control as mean ± SE (n =3).

Fig. 6. Effects of pCMBS on 10 µM ADP-induced adenylyl cyclase inhibition in CHO-K1 cells stably expressing human platelet P2Y12 wild type and mutants. Cells expressing the wild type or mutant P2Y12 receptors were pretreated with pCMBS for 10 min at 37 °C before the addition of 20 µM forskolin. Data were normalized to the value obtained with forskolin alone (mean ± SE. * P > 0.05, *** P < 0.001 compared with forskolin 20 µM + ADP 10 µM by t-test).

28


1 1

MTEVLWPAVPNGTDAAFLAGPGSSWGNSTVASTAAVSSSFKCALTKTGFQFYYLPAVYIL MQAVDNLTSA----------PG----N-----T------SLCTRDYKITQVLFPLLYTVL

P2Y1 P2Y12

61 36

VFIIGFLGNSVAIWMFVFHMKPWSGISVYMFNLALADFLYVLTLPALIFYYFNKTDWIFG FFVGLITNGLAMRIFFQIRSK--SNFIIFLKNTVISDLLMILTFPFKILSDAKLGTGPLR

P2Y1 P2Y12

121 DA---------MCKLQRFIFHVNLYGSILFLTCISAHRYSGVVYPLKSLGRLKKKNAICI 94 TF---------VCQVTSVIFYFTMYISISFLGLITIDRYQKTTRPFKTSNPKNLLGAKIL

P2Y1 P2Y12

172 SVLVWLIVVVAISPILFYSGTGVRKNKTITCYDTTSDEYLRSYFIYSMCTTVAMFCVPLV 145 SVVIWAFMFLLSLPNMILTNRQPRDKNVKKCSFLKSEFGLVWHEIVNYICQVIFWINFLI

P2Y1 P2Y12

232 LILGCYGLIVRALIY-KDLDNSPLRRKSIYLVIIVLTVFAVSYIPFHVMKTMNLRARLDF 205 VIVCYTLITKELYRSYVRTRGVGKVPRKKVNVKVFIIIAVFFICFVPFHFARIPYTLSQT

P2Y1 P2Y12

291 QTPAM-------CAFNDRVYATYQVTRGLASLNSCVDPILYFLAGDTFRRRLSRATRKAS 265 RDVFD-------CTAENTLFYVKESTLWLTSLNACLDPFIYFFLCKSFRNSLISMLKCPN

P2Y1 P2Y12

344 RRSEANLQSKSEDMTLNILPEFKQNGDTSL 318 SATSLSQDNRKKEQDGGDPNEETPM

P2Y1 P2Y12

Fig. 1. Ding et al

29


A

B

Figure 2. Ding et al.

30


120

A

P2Y12WT P2Y12C17S P2Y12C270S P2Y12C17S/C270S

cAMP (%)

100

80

60

40

20

0

0.1

1

10

100

[ADP] µM 120

B


cAMP (%)

100 80 60 40 20

0

0.1

1

[2-MeSADP] nM

Fig. 3. Ding et al.

31

10



140

cAMP (%)

120 100 80 60 40 20 0 -20 0

10

100

[BzATP] µM

Fig. 4. Ding et al.

32

1000


100

A

cAMP (%)

80

60

40

20

0 0

5

10

20

[pCMBS] µM

Fig. 5. Ding et al.

33

Inositol Phosphate formation (% of control)


300

B 250 200 150 100 50 0 1

2

Control

ADP cell#

Fig. 5. Ding et al.

34

3

pCMBS + ADP


120

cAMP (%)

100

forskolin 20 µM forskolin 20 µM + ADP 10 µM forskolin 20 µM + ADP 10 µM + pCMBS 20 µM

*** ***

80

***

60

*

40 20 0

WT (n = 14-15)

C17S (n = 9)

Fig. 6. Ding et al.

35

C270S (n = 11-12)

C17S/C270S (n = 11-12)


Abbreviations. BSA, bovine serum albumin cAMP, 3’,5’-cyclic adenosine monophosphate; P2Y12, platelet ADP receptor coupled to inhibition of adenylyl cyclase; P2Y1, platelet ADP receptor coupled to stimulation of phospholipase C; PLC, Phospholipase C; Gi, heterotrimeric GTP-binding protein which inhibits adenylyl cyclase; Gq, heterotrimeric GTP-binding protein which stimulates phospholipase C; pCMBS, p-chloromercuribenzene sulfonic acid (also known as pCMPS: p -chloromercuriphenyl sulfonic acid); BzATP, 2’-& 3’-O-(4-benzoylbenzoyl) adenosine 5’-triphosphate; 2MeSADP, 2-methylthio-adenosine diphosphate.

36