Arg333 and Arg334 in the COOH terminus of the human P2Y1 receptor are crucial for Gq coupling

Zhongren Ding,1,3 Florin Tuluc,1 Kavita R. Bandivadekar,1 Lili Zhang,1 Jianguo Jin,1 and Satya P. Kunapuli1,2,3

1Department of Physiology, 2Department of Pharmacology, and 3The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania

Submitted 16 August 2004 ; accepted in final form 20 October 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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The P2Y1 ADP receptor activates Gq and causes increases in intracellular Ca2+ concentration through stimulation of PLC. In this study, we investigated the role of the amino acid residues in the COOH terminus of the human P2Y1 receptor in Gq activation. Stimulation of Chinese hamster ovary (CHO-K1) cells stably expressing the wild-type human P2Y1 receptor (P2Y1-WT cells), P2Y1-{Delta}R340-L373, or P2Y1-{Delta}D356-L373 with 2-methylthio-ADP (2-MeSADP) caused inositol phosphate production. In contrast, cells expressing P2Y1-{Delta}T330-L373, a mutant lacking the entire COOH terminus, completely lost their response to 2-MeSADP. Similar data were obtained by using these cell lines and measuring Ca2+ mobilization upon stimulation with 2-MeSADP, indicating that the 10 amino acids (330TFRRRLSRAT339) in the COOH terminus of the human P2Y1 receptor are essential for Gq coupling. Radioligand binding demonstrated that both the P2Y1-WT and P2Y1-{Delta}T330-L373-expressing cells have almost equal binding of [3H]MRS2279, a P2Y1 receptor antagonist, indicating that COOH-terminal truncation did not drastically affect the conformation of the receptor. CHO-K1 cells expressing a chimeric P2Y12 receptor with the P2Y1 COOH terminus failed to elicit Gq functional responses, indicating that the P2Y1 COOH terminus is essential but not sufficient for Gq activation. Finally, cells expressing a double-mutant P2Y1 receptor (R333A/R334A) in the conserved BBXXB region of the COOH terminus of the Gq-activating P2Y receptors completely lost their functional ability to activate Gq. We conclude that the two arginine residues (R333R334) in the COOH terminus of the human P2Y1 receptor are essential for Gq coupling.

carboxyl terminus; adenosine diphosphate; truncation; inositol phosphate


EXTRACELLULAR NUCLEOTIDES influence many biological functions, including vascular tone, cell division, cardiac and skeletal muscle contraction, and platelet aggregation, as well as peripheral and central neurotransmission (7). Extracellular nucleotides can trigger intracellular effects by specifically binding to and activating cell surface membrane proteins known as P2 receptors (7, 14, 15, 18). Two main families of receptors for extracellular nucleotides have been described: P2X receptors, which are ligand-gated ion channels, and P2Y receptors, which belong to the superfamily of G protein-coupled receptors (7). Nine distinct P2Y receptors are expressed in human tissues: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14, and P2Y15 (1, 7, 14, 27). However, the P2Y15 receptor might indeed be a receptor for {alpha}-ketoglutarate (23).

The human P2Y1 receptor was originally cloned from human erythroleukemia cells (4) and endothelial cells (37). The human P2Y1 receptor is 373 amino acids long with 7 putative hydrophobic transmembrane regions and is encoded by a single exon (3). The gene for the human P2Y1 receptor has been localized to chromosome 3q25 (3). When heterologously expressed in astrocytoma cells, the P2Y1 receptor has been shown to activate PLC through pertussis toxin-insensitive G proteins of the Gq/11 class, resulting in inositol phosphate formation (44). Studies with mice lacking Gq revealed that signaling through Gq is essential for ADP-induced functional responses in platelets (40).

ADP and 2-methylthio-ADP (2-MeSADP) are potent agonists at the P2Y1 receptor, whereas adenosine 3'-phosphate 5'-phosphosulfate, adenosine 3'-phosphate 5'-phosphate, and adenosine 2'-phosphate 5'-phosphate are selective antagonists (6). Mutational analysis studies have been performed to locate the essential residues for ligand recognition, identifying several amino acids in the extracellular loops and transmembrane domains that were essential for ligand recognition (25, 28).

P2Y1 was the first P2Y receptor to be cloned and has a wide distribution, having been described in heart, vascular, connective, immune, and neural tissues (43). P2Y1 receptors may be involved in the modulation of neurotransmission (48). However, the function of the P2Y1 receptor has been studied most extensively in platelets (33). The P2Y1 receptor plays an important role in ADP-induced shape change (29), aggregation (30), and thromboxane A2 (TXA2) generation (31). P2Y1 receptor-deficient mice have increased bleeding times and are protected from thromboembolism (19, 36). These results suggest that the P2Y1 signaling blockade, at the receptor-G protein interaction level, might be a potential antithrombotic drug target. With this goal in mind, we initiated studies to elucidate the Gq coupling domains in the P2Y1 receptor.

In this study, we first sought to explore the role of the COOH-terminal region of human P2Y1 receptor in receptor-mediated Gq activation and found that the COOH terminus is critical for the Gq coupling ability of the receptor. Furthermore, we also identified two arginine residues (R333R334) in the COOH-terminal region that are essential for Gq coupling.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Materials. 2-MeSADP was obtained from Sigma Chemical (St. Louis, MO). Myo-2-[3H]inositol was obtained from NEN Life Science (Boston, MA). [3H]MRS2279 was used in the laboratory of Dr. T. Kendall Harden (Dept. of Pharmacology, Univ. of North Carolina School of Medicine, Chapel Hill, NC). All oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). FITC-labeled monoclonal antibody (HA.11) against HA (hemagglutinin epitope tag) was purchased from Covance Research Products (Berkeley, CA).

Construction of human P2Y1 truncation mutant plasmids. Human platelet P2Y1 receptor (GenBank accession no. U42029) (3) was cloned into the pcDNA3 vector with a HA tag (YPYDVPDYA) inserted at the beginning of the translation initiation by polymerase chain reaction (PCR). Forward primer containing a BamHI restriction site (underlined) and a HA tag sequence was 5'-GCGGATCCACCATGTACCCATACGATGTTCCAGATTACGCTACCGAGGTGCTGTGGCCGGCT-3'. Three COOH-terminally truncated P2Y1 receptors (P2Y1-{Delta}T330-L373, P2Y1-{Delta}R340-L373, and P2Y1-{Delta}D356-L373) were constructed using PCR with the same forward primer (as for wild-type P2Y1), a reverse primer with a stop codon at the desired location (Fig. 1 and Table 1), and an XhoI restriction site to aid in subcloning. All truncated receptors were subcloned into pcDNA3 and verified using DNA sequence analysis.



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Fig. 1. Depiction of COOH terminus sequence and the truncations of human P2Y1 receptor. Three COOH-terminally truncated P2Y1 receptors, P2Y1-{Delta}T330-L373, P2Y1-{Delta}R340-L373, and P2Y1-{Delta}D356-L373, were constructed to be expressed in Chinese hamster ovary (CHO-K1) cells. Arrows show the locations of truncation mutations introduced into the COOH terminus of the P2Y1 receptor.

 

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Table 1. Reverse primers for human P2Y1 receptor COOH-terminal truncation mutation

 
Construction of human P2Y12/P2Y1 chimeric receptor expression plasmid. One human P2Y12/P2Y1 chimera was constructed using overlap-extension PCR with the COOH terminus of human platelet P2Y12 receptor (GenBank accession no. AF313449) (26) replaced by human P2Y1 receptor (GenBank accession no. U42029). With the use of human P2Y12 in pcDNA3.1/Hygro(+) (13) as template, the first part of the chimera was PCR amplified using forward primer containing a KpnI restriction site (underlined) and the HA tag sequence (designated primer I), 5'-GCGCGGTACCACCATGTACCCATACGATGTTCCAGATTACGCTCAAGCCGTCGACAATCTC-3'; the reverse primer that matched both parts (designated primer II) was 5'-AGTCTCCTTCTGAAAGTCTTGCAAAGGAAAAAATA-3'. For the second part of the chimera, human platelet P2Y1 receptor in pcDNA3 was used as template. The forward primer that matched both parts (designated primer III) was 5'-ATTTTTTCCTTTGCAAGACTTTCAGAAGGAGACTC-3', and the reverse primer containing a XhoI restriction site (designated primer IV) was 5'-GCCTCGAGCAGGCTTGTATCTCCATTCTGC-3'. After confirmation that the two parts were successfully amplified, 1 µl of each part used as template was added in a 50-µl reaction volume, and the second cycle of PCR was performed by using primer I as forward primer and primer IV as reverse primer to amplify the full length of the chimera P2Y12/P2Y1. The overlap-extension PCR-amplified P2Y12/P2Y1 was then cloned into pcDNA3 by digesting the PCR product with KpnI and XhoI and inserted into the vector digested with the same set of restriction enzymes. The nucleotide sequence of the P2Y12/P2Y1 receptor coding sequence in the expression plasmid was confirmed using DNA sequence analysis.

Construction of P2Y1-R333A/R334A expression plasmid. Human P2Y1-R333A/R334A expression plasmid was constructed using wild-type human P2Y1 receptor expression plasmid as template by performing site-directed mutagenesis as described previously (13), using forward primer 5'-GGGAGATACTTTCAGAGCGGCACTCTCCCGAGCCACAAGG-3' and reverse primer 5'-CCTTGTGGCTCGGGAGAGTGCCGCTCTGAAAGTATCTCCC-3'. The nucleotide sequence of the P2Y1-R333A/R334A encoding sequence in the expression plasmid [P2Y1-R333A/R334A-pcDNA3/G418(+)] was confirmed using DNA sequence analysis.

Cell culture. Chinese hamster ovary (CHO-K1) cells were grown in Ham's F-12 medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum and 1% penicillin, streptomycin, and amphotericin B at 37°C with 5% CO2. CHO-K1 cells transfected with wild-type or mutant P2Y1 receptors were grown in the same medium supplemented with 500 µg/ml G418.

Stable expression of human P2Y1 receptor in CHO-K1 cells. The expression construct for the wild-type P2Y1 receptor or for each of the P2Y1 mutants (1 µg) was used to transfect CHO-K1 cells by using Lipofectamine as described previously (2). After 6 h, the growth medium was replaced with fresh medium. Stable transfectants were selected on medium containing 500 µg/ml G418 and were screened for the expression of wild-type or mutant P2Y1 receptor by HA tag detection using flow cytometry.

HA tag detection using flow cytometry. CHO-K1 cells, naive, vector transfected, or stably transfected with P2Y1 receptors, were cultured in 100-mm dishes, washed twice with PBS (137 mM NaCl, 2.68 mM KCl, 4.29 mM Na2HPO4, and 1.47 mM KH2PO4) and detached with Versene (0.5 mM Na4EDTA, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 15 mM KH2PO4, and 1 mM glucose). After spinning at 700 rpm for 3 min, the pellets were resuspended in Tyrode solution [137 mM NaCl, 2.67 mM KCl, 2 mM MgCl2, 2.03 mM NaH2PO4, 5.6 mM glucose, 10 mM HEPES, and 0.2% bovine serum albumin (BSA), pH 7.4] and cell concentrations were adjusted to 107 cells/ml. Aliquots of 100 µl of cell suspension were mixed with 4 µl of 1:10 diluted FITC-labeled monoclonal antibody against HA (Covance) in the presence of 2 mM Ca2+. After incubation at 4°C for 1 h in the dark, cell suspensions were briefly spun, and the supernatant was discarded. Cells were resuspended in 400 µl of Tyrode solution and analyzed using flow cytometry with FACScan (BD Biosciences). Untransfected CHO-K1 cells were used as negative control.

Measurement of inositol phosphate levels. Inositol phosphate levels were measured as described previously with minor modifications (13, 20, 32). Confluent cultures of cells in 12-well plates were labeled with 1 µCi/ml (0.037 MBq/ml) myo-2-[3H]inositol in inositol-free DMEM for 16–24 h. Labeled cells were washed once, the medium was replaced with 970 µl of fresh inositol-free DMEM containing 20 µl of 1 M LiCl, and the cells were incubated at 37°C for 15 min. The cells were then stimulated with 10 µl of 2-MeSADP (varying concentrations) for 10 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 45 min. The solution containing the extracted inositol phosphates was neutralized by dilution with 3 ml of 5 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 (Bio-Rad, Hercules, CA), 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. The eluate (1 ml) was removed and counted with 9 ml of scintillation fluid.

Measurement of cytoplasmic free Ca2+ concentration. Cultured cells were harvested and incubated for 45 min at room temperature in Hanks' buffered salt solution (HBSS; Mediatech) containing 1 µM fura-2 AM (Molecular Probes, Eugene, OR) and 0.2% BSA. Cells were washed twice and then resuspended in HBSS containing 0.2% BSA, and cytoplasmic free Ca2+ concentration was measured as previously described (46). Briefly, aliquots of 0.7 ml of cell suspension were placed in disposable methacrylate cuvettes under stirring conditions at 37°C. Fluorescence was recorded on an Aminco-Bowman spectrofluorometer (Spectronics Instruments, Rochester, NY) during agonist stimulation by using alternating excitation wavelengths of 340 and 380 nm and monitoring emitted light at 510 nm. Cytoplasmic concentrations of Ca2+ were calculated according to Tsien's ratiometric method (22).

Radioligand binding assay. Binding of the high-affinity P2Y1 receptor antagonist (5) [3H]MRS2279 to wild-type or truncated P2Y1 receptors was performed as described elsewhere (49). Briefly, cell membranes (125 µg of protein) were incubated at 4°C with 30 nM [3H]MRS2279 (~120,000 cpm) in 20 mM Tris, pH 7.5, 145 mM NaCl, and 5 mM MgCl2 in a volume of 50 µl. Specific binding was usually defined as total [3H]MRS2279 binding minus binding occurring in the presence of a 10 µM concentration of the P2Y1 receptor-specific antagonist MRS2179. Incubations proceeded for 30 min at 4°C. Binding reactions were terminated by addition of 4 ml of ice-cold wash buffer (10 mM Tris, pH 7.5 at 4°C, and 145 mM NaCl) and rapid filtration over GF/A glass fiber filters. Each filter was washed with an additional 4 ml of ice-cold wash buffer. Radioactivity was quantitated using liquid scintillation spectrometry.

Statistical analysis. Data are expressed as means ± SE. Statistical significance was determined using Student's t-test and was designated at P values <0.05.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Stable expression of P2Y1 wild-type and COOH-terminal truncations in CHO-K1. Different constructs of the human P2Y1 receptor (outlined in Fig. 1) were prepared as described in MATERIALS AND METHODS. CHO-K1 cells were transfected with the P2Y1 wild-type receptor and mutants, and clones resistant to 500 µg/ml G418 were selected. We screened the clones on the basis of HA tag expression on the cell surface as detected by flow cytometry using a FITC-labeled monoclonal antibody against an HA tag located at the NH2 terminus of the receptor. Transfected cells were cultured in the presence of 500 µg/ml G418 for 14 days, and then cells expressing the HA tag were sorted using a Cytomation MoFlo (Fort Collins, CO) before proceeding with the single-cell cloning process. The successful expression of the wild-type and mutant P2Y1 receptors in CHO-K1 cells was confirmed by HA tag detection using flow cytometry (Fig. 2A and Table 2). All cell lines expressing COOH-terminally truncated P2Y1 receptors showed equal or higher levels of expression of the HA tag compared with cells expressing the wild-type P2Y1 receptor.



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Fig. 2. Intracellular responses of the wild-type (WT) and truncated P2Y1 receptors. A: total inositol phosphate (IP) measurements. B: intracellular Ca2+ mobilization. CHO-K1 cells ({circ}) and CHO-K1 cells expressing the P2Y1-WT ({bullet}) or P2Y1-{Delta}T330-L373 ({blacksquare}) mutants were exposed to varying concentrations of 2-methylthio-ADP (2-MeSADP), and the intracellular responses were measured as described in MATERIALS AND METHODS. IP generation is expressed as counts per minute (cpm). Results are expressed as means ± SE (n = 4–8). Inset shows flow cytometry analysis of surface expression of hemagglutinin (HA)-tagged human P2Y1 receptor in CHO-K1 cells. CHO-K1 cells or CHO-K1 cells transfected with P2Y1-WT or P2Y1-{Delta}T330-L373 receptors were incubated at 4°C for 1 h in the dark with anti-HA FITC-labeled monoclonal antibody. Cell suspensions were briefly spun, and the pellets were resuspended in 400 µl of Tyrode solution and analyzed using flow cytometry. Representative results are shown for 2–7 separate experiments.

 

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Table 2. 2-MeSADP-stimulated PLC activation and surface expression assays (flow cytometry) of wild-type and truncated human P2Y1 receptors

 
COOH-terminal truncation abolished Gq activation by P2Y1 receptor. 2-MeSADP, a human platelet P2Y1 receptor agonist, concentration-dependently activated the wild-type P2Y1 receptor (P2Y1-WT) that was stably expressed in CHO-K1 cells and resulted in the production of inositol phosphates (Fig. 2A) and intracellular Ca2+ mobilization (Fig. 2B). P2Y1-{Delta}T330-L373 stably expressed in CHO-K1 cells did not respond to 2-MeSADP up to 1 µM in concentration (Fig. 2, A and B). The EC50 values for inositol phosphate production and for intracellular Ca2+ mobilization are summarized in Table 2. We determined that the COOH-terminal truncation did not affect receptor expression on the surface of CHO-K1 cells (Fig. 2A and Table 2). Hence, abolished cellular responses in P2Y1 deletion mutant in response to 2-MeSADP are not due to low expression levels of the P2Y1 receptor and are likely due to loss of an important domain necessary for coupling to Gq.

[3H]MRS2279 binds to both wild-type and COOH-terminally truncated P2Y1 receptors. To confirm that the COOH terminus truncation did not drastically alter the conformation and thereby interfere with the ligand binding ability, we measured the binding of [3H]MRS2279 to cell membranes derived from CHO-K1 cells expressing P2Y1-WT or P2Y1-{Delta}T330-L373. As shown in Fig. 3, the binding of [3H]MRS2279 to the truncated receptor was not significantly different from the binding to the wild-type receptor. On the basis of these results, we conclude that truncation at the COOH-terminal domain did not result in altered folding of the P2Y1 receptor or lower the affinity of ligands for the receptor. Hence, the inability of the truncated P2Y1 receptor to trigger intracellular signaling is caused by the impaired interaction with the Gq protein. We therefore propose that the human platelet P2Y1 receptor COOH terminus is a Gq-coupling region that is crucial for the interaction between Gq and the P2Y1 receptor.



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Fig. 3. Binding of [3H]MRS2279 to CHO-K1 cells stably expressing human P2Y1-WT or P2Y1-{Delta}T330-L373 receptors. Cell membranes were incubated with [3H]MRS2279 at 4°C for 30 min. Specific binding was defined as total [3H]MRS2279 binding minus binding occurring in the presence of a 10 µM concentration of MRS2179, a different P2Y1 receptor antagonist. Bars represent means ± SE of data obtained from experiments performed in duplicate (n = 3).

 
P2Y1 receptor COOH terminus is not sufficient for Gq activation. Because deletion of the COOH terminus of the P2Y1 receptor resulted in the complete loss of Gq activation, we investigated whether this domain is the Gq coupling domain in the P2Y1 receptor. We achieved this by constructing an expression plasmid for a chimeric P2Y12 receptor in which the coding sequence for the COOH terminus was replaced with that for the human P2Y1 receptor (Fig. 4A). The P2Y12 receptor has the same agonist profile as the P2Y1 receptor and activates Gi but not Gq (33). We reasoned that if the COOH terminus of the P2Y1 receptor is sufficient for activation of Gq, then the chimeric P2Y12/P2Y1 should stimulate PLC upon stimulation with ADP. As shown in Fig. 4B, inset, the chimeric receptor is expressed on the surface as detected by HA tag in CHO-K1 cells transfected with the expression plasmid. However, these cells stably expressing the chimeric receptor failed to stimulate Gq, as evidenced by a lack of increases in inositol phosphate generation (Fig. 4B) or increased intracellular Ca2+ mobilization (not shown). The chimeric P2Y12/P2Y1 receptor still retained the Gi stimulating ability with an EC50 value similar to that of wild-type P2Y12 when 2-MeSADP-induced adenylyl cyclase was measured (data not shown).



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Fig. 4. Expression and functional analysis of the P2Y12/P2Y1 chimeric receptor. A: schematic representation of the construction of the P2Y12/P2Y1 chimeric receptor. B: CHO-K1 cells expressing the P2Y1-WT receptor or the P2Y12/P2Y1 chimeric receptor were exposed to 1 µM 2-MeSADP (filled bars), and IP generation was measured as described in MATERIALS AND METHODS. IP formation in unstimulated cells is represented by open bars. Results are expressed as means ± SE (n = 4–8). Inset shows surface expression of the chimeric receptor detected using the HA tag as described in Fig. 2.

 
COOH-terminal truncation at R340 and D356 did not abolish coupling of P2Y1 to Gq protein. The COOH terminus of the human P2Y1 receptor is composed of 44 amino acids, and this region appears to be critical for coupling to Gq. To more precisely locate the specific domain responsible for Gq coupling, we specifically truncated the COOH terminus of P2Y1 at different sites and constructed two additional human platelet P2Y1 truncations, P2Y1-{Delta}R340-L373 and P2Y1-{Delta}D356-L373 (see Fig. 1), which were expressed in CHO-K1 cells. The successful expression of these two truncated P2Y1 receptors was confirmed by detection of the HA tag on the surface of CHO-K1 cells (Fig. 5, inset, and Table 2). The function of these two truncations was evaluated by assaying the production of inositol phosphate and the mobilization of intracellular Ca2+ upon stimulation with 2-MeSADP. In contrast to its effect in the cell line expressing the P2Y1-{Delta}T330-L373 receptor, 2-MeSADP concentration-dependently activated PLC in CHO-K1 cells stably expressing P2Y1-{Delta}R340-L373 and P2Y1-{Delta}D356-L373 (Fig. 5, A and B). The concentration-response curves for the mutants were shifted slightly to the right, and the corresponding EC50 values for production of inositol phosphate (Fig. 5A) and Ca2+ mobilization (Fig. 5B) were slightly higher compared with those for the cell line expressing the P2Y1-WT receptor (Table 2). These results suggest that the domain between R340 and L373 is not essential in conferring the Gq-coupling ability to the P2Y1 receptor. We therefore conclude that the 10-amino acid segment between T330 and T339 (TFRRRLSRAT) in the COOH terminus of the P2Y1 receptor is crucial for coupling to the Gq protein.



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Fig. 5. Effects of COOH-terminal truncations on the function of human platelet P2Y1 in CHO-K1 cells. CHO-K1 cells expressing the P2Y1-WT receptor ({bullet}), P2Y1-{Delta}R340-L373 mutant ({circ}), or P2Y1-{Delta}D356-L373 mutant ({square}) were exposed to varying concentrations of 2-MeSADP, and the intracellular responses were measured as described in MATERIALS AND METHODS. A: IP generation. B: intracellular Ca2+ values were normalized to the maximum response elicited by 2-MeSADP in CHO-K1 cells expressing the P2Y1-WT receptor, which was considered 100%. Results are expressed as means ± SE (n = 4–8). Inset shows flow cytometry analysis of surface expression of HA-tagged human P2Y1 receptor in CHO-K1 cells. CHO-K1 cells or CHO-K1 cells transfected with P2Y1-{Delta}R340-L373 or P2Y1-{Delta}D356-L373 receptors were analyzed using flow cytometry. Representative results are shown for 2–7 separate experiments.

 
Previous studies have shown that the COOH terminus of the P2Y1 receptor also regulates the inactivation gating of an ion channel in oocytes (35). The domain involved in this interaction has been delineated by deletion mutagenesis (35). These studies identified a short region, R340-L359, in the COOH terminus of the P2Y1 receptor interacting with this ion channel (35). In contrast, deletion of R340 to L373 did not affect the Gq-activating ability of the P2Y1 receptor (Fig. 5). Thus the domains interacting with the oocyte ion channel and the Gq protein are distinct in the COOH terminus of the P2Y1 receptor.

Comparison of the COOH-terminal sequence of the human P2Y1 receptor with the P2Y1 receptor COOH-terminal sequence of other species revealed regions of high consensus (Fig. 6A). The human P2Y1 COOH terminus has 97.7% identity with that of bovine and guinea pig, 95.4% identity with that of rat and mouse, and 86.3% with that of chicken. Among the three domains of human P2Y1 receptor COOH terminus investigated in this study (T330-T339, R340-E355, and D356-L373), only the first 10 amino acids in the T330-T339 domain of P2Y1 COOH terminus for human and the other five species (bovine, guinea pig, rat, mouse, and chicken) are 100% identical. The high consensus of amino acids of this domain may be related to the requirement of this domain in Gq activation. It is noteworthy that this region (T330-T339) also contains the BBXXB motif (where B is a basic residue and X is a non-basic residue) that is frequently involved in Gq coupling (34). The two consecutive arginine residues of the BBXXB also exist in the other Gq-coupled human P2Y receptors such as P2Y4 and P2Y6 (Fig. 6B). This implies that these two arginine residues, Arg333 and Arg334, may be critically important for P2Y receptor-mediated Gq activation.



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Fig. 6. Comparison of the COOH-terminal sequence of the P2Y1 receptor from different species (A) and of different P2Y receptors (B). A sequence of 10 amino acids in the T330-T339 domain of P2Y1 COOH terminus is conserved in human and 5 other species (bovine, guinea pig, rat, mouse, chicken) (11, 12, 24, 42, 45, 50). The high consensus of amino acids may be related to the requirement of this domain for Gq activation. Amino acids that are different among species are shown in bold type (A). The 10-amino acid sequence T330-T339 is not conserved in other human P2Y receptors. The putative Gq-interacting motif B-B-X-X-B is shown in bold type (B). The total number of amino acids in each P2Y receptor is shown at right (B).

 
Role of Arg333 and Arg334 residues in the P2Y1 receptor in Gq coupling. Site-directed mutagenesis was performed to determine whether these two arginine residues are indeed critical for recognition of Gq by P2Y1 receptor. A double mutant of human P2Y1 receptor plasmid was generated wherein these two arginine residues were mutated to alanine. The construct was transfected into CHO-K1 cells, and two clones expressing the receptor were selected. As shown in Fig. 7A, inset, the D3 clone is expressed at lower levels than the wild-type receptor in the P2Y1-WT clone, whereas the G12 clone is expressed at higher levels. The functional evaluation was performed in these cells upon stimulation with 2-MeSADP. As shown in Fig. 7A, neither of these double-mutant P2Y1 receptor-expressing cells caused any inositol phosphate generation in response to 2-MeSADP. Similarly, 2-MeSADP failed to cause any mobilization of Ca2+ from intracellular stores in these cells expressing the double-mutant P2Y1 receptors (Fig. 7B). These data demonstrate the importance of the Arg333 and Arg334 residues in Gq activation by the P2Y1 receptor.



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Fig. 7. Effects of the double arginine mutation to alanine (R333A/R334A) on the function of human P2Y1 in CHO-K1 cells. CHO-K1 cells expressing the P2Y1-WT receptor, the P2Y1-R333A/R334A double-mutant clone D3 (low-level expression), or clone G12 (high-level expression) were exposed to 1 µM 2-MeSADP, and the intracellular responses were measured as described in MATERIALS AND METHODS. A: IP generation was normalized to the base level in unstimulated cells. B: intracellular Ca2+ levels ([Ca2+]i). Results are expressed as means ± SE (n = 3). Inset shows flow cytometric analysis of surface expression of HA-tagged human P2Y1 receptor R333A/R334A mutant in CHO-K1 cells. CHO-K1 cells, 2 separate clones of CHO-K1 cells expressing P2Y1-R333A/R334A mutant receptors, or P2Y1-WT were analyzed using flow cytometry. Representative results are shown for 2–4 separate experiments.

 
The P2Y2 receptor was originally known as the P2U receptor and was shown to couple to pertussis toxin-sensitive G proteins (15–18, 42). However, the presence of BBXXB sequence in the COOH terminus of the P2Y2 receptor (highlighted in bold in Fig. 6B) suggests that this receptor might activate pertussis toxin-insensitive G proteins as well. Thus the P2Y2 receptor might couple to both pertussis toxin-sensitive and -insensitive G proteins. Consistent with this notion, pertussis toxin only partially inhibits P2Y2 receptor-mediated intracellular Ca2+ mobilization (38, 42). Previously, Weisman and coworkers (21, 41) generated truncated mutants of the P2Y2 receptor and studied their ability to be desensitized. These truncations in the COOH terminus of the P2Y2 receptor are similar to P2Y1-{Delta}R340-L373 and P2Y1-{Delta}D356-L373. Similar to the P2Y1 truncations, the P2Y2 truncations in the COOH terminus retained their ability to couple to the G proteins. However, these investigators have not deleted the HRPNR (BBXXB) from the murine P2Y2 receptor, and hence it is not clear whether these amino acids in the P2Y2 receptor are also important for Gq protein coupling (21, 41). Future studies are needed to evaluate these possibilities.

The P2Y12, P2Y13, and P2Y14 receptors couple to pertussis toxin-sensitive G proteins (8, 26, 52, 53). The functional effects of these three receptors are abolished by pertussis toxin treatment (8, 26, 53). These data and the lack of BBXXB motif in the COOH termini of these receptors (Fig. 6B) are consistent with their inability to activate the Gq class of proteins. Finally, the P2Y11 and P2Y15 receptors couple to both Gq and Gs proteins (10, 27) but lack the BBXXB motif in their COOH termini (Fig. 6B). It is possible that these two receptors use some other motif for Gq coupling.

The BB motif in BBXXB is found not only in P2Y but also in other Gq-coupled receptors. Human TXA2 receptor contains two consecutive arginine residues at the juxtamembrane COOH terminus (47). Moreover, three human {alpha}1A-adrenergic receptor isoforms ({alpha}1A-1, {alpha}1A-2, and {alpha}1A-3) (9) as well as the human platelet-activating factor receptor contain two consecutive basic amino acids (KK) at the juxtamembrane position in the COOH terminus (39). However, it is noteworthy that the protease-activated receptor 4 lacks the doublet of basic amino acids in the COOH terminus (51), suggesting that the BB motif is not always required for Gq coupling.

In conclusion, we have shown that the Arg333 and Arg334 residues in the COOH terminus of the P2Y1 receptor are important for the G protein coupling. This is the first report of the identification of an important region and residues in the P2 receptor family for G protein coupling.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-60683, HL-64943, and HL-63933 (to S. P. Kunapuli).


    ACKNOWLEDGMENTS
 
We thank Dr. T. Kendall Harden (Dept. of Pharmacology, Univ. of North Carolina School of Medicine, Chapel Hill, NC) for providing [3H]MRS2279 and Savitri Madiletti (Dept. of Pharmacology, Univ. of North Carolina School of Medicine, Chapel Hill, NC) for the radioligand binding assay. We also thank Drs. Todd M. Quinton and Robert T. Dorsam (Sol Sherry Thrombosis Research Center, Temple Univ. School of Medicine, Philadelphia, PA) for critically reviewing the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. P. Kunapuli, Dept. of Physiology, Temple Univ. School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140 (E-mail: spk{at}temple.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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