Abstract
The P2Y14 receptor was initially identified as a G protein-coupled receptor activated by UDP-glucose and other nucleotide sugars. We have developed several cell lines that stably express the human P2Y14 receptor, allowing facile examination of its coupling to native Gi family G proteins and their associated downstream signaling pathways (J Pharmacol Exp Ther 330:162–168, 2009). In the current study, we examined P2Y14 receptor-dependent inhibition of cyclic AMP accumulation in human embryonic kidney (HEK) 293, C6 glioma, and Chinese hamster ovary (CHO) cells stably expressing this receptor. Not only was the human P2Y14 receptor activated by UDP-glucose, but it also was activated by UDP. The apparent efficacies of UDP and UDP-glucose were similar, and the EC50 values (74, 33, and 29 nM) for UDP-dependent activation of the P2Y14 receptor in HEK293, CHO, and C6 glioma cells, respectively, were similar to the EC50 values (323, 132, and 72 nM) observed for UDP-glucose. UDP and UDP-glucose also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y14 receptor-expressing HEK293 cells but not in wild-type HEK293 cells. A series of analogs of UDP were potent P2Y14 receptor agonists, but the naturally occurring nucleoside diphosphates, CDP, GDP, and ADP exhibited agonist potencies over 100-fold less than that observed with UDP. Two UDP analogs were identified that selectively activate the P2Y14 receptor over the UDP-activated P2Y6 receptor, and these molecules stimulated phosphorylation of ERK1/2 in differentiated human HL-60 promyeloleukemia cells, which natively express the P2Y14 receptor but had no effect in wild-type HL-60 cells, which do not express the receptor. We conclude that UDP is an important cognate agonist of the human P2Y14 receptor.
The metabotropic P2Y receptors include a subgroup of five receptors, the P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors, that primarily signal through Gq-activated signaling pathways and a subgroup of three receptors, the P2Y12, P2Y13, and P2Y14 receptors, that primarily signal by activating heterotrimeric G proteins of the Gi family (Abbracchio et al., 2006; Burnstock, 2007). The human P2Y1, P2Y11, P2Y12, and P2Y13 receptors are activated by adenine nucleotides. The human P2Y4 and P2Y6 receptors are activated by uridine nucleotides, and the P2Y2 receptor is activated by both ATP and UTP.
The P2Y14-R exhibits the most unique agonist selectivity of the P2Y receptors extant; it was initially identified as an orphan G protein-coupled receptor that is activated by nucleotide sugars, such as UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, and UDP-N-glucuronic acid, rather than by adenine and/or uridine nucleotides (Chambers et al., 2000; Freeman et al., 2001). Although the P2Y14-R has been considered a nucleotide sugar-regulated receptor, we recently reported that UDP is a competitive antagonist/partial agonist at the human ortholog (Fricks et al., 2008). Moreover, our experiments with the rat P2Y14-R suggested that UDP is a potent full agonist at this receptor. These studies revealing activities of UDP were carried out in COS-7 cells transiently coexpressing recombinant P2Y14-R with a chimeric G protein, Gαq/i (Coward et al., 1999) that couples Gi-activating receptors to activation of phospholipase C-β isozymes. Thus, we questioned whether the action of UDP previously observed by measuring inositol lipid hydrolysis was a vagary of the test system used. To address this issue, agonist activities were quantified in three different stable cell lines we developed that allow study of P2Y14-R-dependent inhibition of adenylyl cyclase activity. These experiments measuring a natural cell signaling response of the P2Y14-R reveal that not only is UDP an agonist of the human receptor, but its potency and apparent efficacy are similar to that of UDP-glucose and other nucleotide sugars. Thus, the pervasive view that the P2Y14-R is selectively activated by UDP-glucose and other nucleotide sugars should be reassessed with the idea that extracellular UDP is also an important cognate agonist of this receptor.
Materials and Methods
Cell Lines Stably Expressing P2Y14-R.
Human embryonic kidney 293 cells stably expressing the hP2Y14-R (P2Y14-HEK293 cells) and C6 rat glioma cells stably expressing the hP2Y14-R (P2Y14-C6 cells) were generated as described previously (Fricks et al., 2009). Chinese hamster ovary cells stably expressing the hP2Y14-R (P2Y14-CHO cells) were produced using similar methods. In brief, hP2Y14-R cDNA was ligated into the retroviral expression vector pLXSN as described previously (Wolff et al., 2005), and recombinant retrovirus for hP2Y14-R expression was produced in PA317 cells. CHO cells were incubated with hP2Y14-R virus and 0.4 mg/ml G418 for 2 weeks to produce hP2Y14-R-expressing cells.
Cell Culture.
HEK293 cells were grown in DMEM supplemented with 10% fetal bovine serum and 1% G418 (Geneticin) at 37°C in a 5% CO2 environment. C6 rat glioma cells were cultured in DMEM supplemented with 5% fetal bovine serum and 1% G418 in a 5% CO2 environment. CHO cells were grown in Ham's F-12 medium supplemented with 10% fetal bovine serum and 1% G418 at 37°C in a 5% CO2 environment. 1321N1 Human astrocytoma cells were grown in DMEM supplemented with 5% fetal bovine serum in a 5% CO2 environment. HL-60 promyeloleukemia cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. Differentiation of HL-60 cells was achieved by inclusion of 1.3% DMSO in the culture medium for 5 days.
Cyclic AMP Accumulation.
Cells were grown in 24-well plates and incubated with 1 μCi of [3H]adenine/well in serum-free DMEM for 2 h before assay. Assays were initiated by the addition of HEPES-buffered, serum-free DMEM containing 200 μM 3-isobutyl-1-methylxanthine and agonists, and incubation continued for 10 min at 37°C. Incubations were terminated by aspiration of medium and addition of 500 μl of ice-cold 5% trichloroacetic acid. [3H]cAMP was isolated by sequential Dowex and alumina chromatography and quantified by liquid scintillation counting as described previously (Harden et al., 1982).
Inostiol Phosphate Accumulation.
P2Y6-R-dependent activation of phospholipase C was measured by quantifying [3H]inositol phosphate accumulation in 1321N1 human astrocytoma cells as described previously (Besada et al., 2006).
MAP Kinase Activation Assays.
Phosphorylation of ERK1/2 was quantified as we have described in detail (Fricks et al., 2009). In brief, wild-type HEK293 or P2Y14-HEK293 cells were serum-starved for 18 h before addition of either UDP or UDP-glucose for 15 min at 37°C. Undifferentiated or differentiated HL-60 cells were serum-starved for 18 h before addition of agonist for 30 min at 37°C. Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis on a 12.5% polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane, which was sequentially incubated with antibody for phospho-ERK1/2, with horseradish peroxidase-conjugated goat anti-mouse antibody, and with chemiluminescent substrate (Pierce West Pico system; Thermo Fisher Scientific, Waltham, MA) and then was exposed to film. Membranes were stripped and reprobed with a primary antibody against total ERK1/2 to verify equal loading of lanes.
Data Analysis.
EC50 values were determined using Prism software (GraphPad, San Diego, CA) and are presented as mean ± S.E. All experiments were repeated at least three times.
Materials.
3-Isobutyl-1-methylxanthine and forskolin were purchased from SigmaAldrich (St. Louis, MO). UDP-glucose, UDP, CDP, GDP, and ADP were all from FLUKA and were purchased from SigmaAldrich. High-performance liquid chromatography analysis of UDP-glucose and all of the nucleotides revealed >95% purity. Pertussis toxin was purchased from List Biologicals (Campbell, CA). [3H]Adenine was purchased from American Radiolabeled Chemicals (St. Louis, MO). G418, serum, and all cell culture medium were from Invitrogen (Carlsbad, CA). Antibodies for phosphoERK1/2 and ERK1/2 were purchased from Cell Signaling Technologies (Danvers, MA).
Results
Agonist-promoted activation of Gi and consequential inhibition of adenylyl cyclase is thought to be a primary signaling response of the P2Y14-R and other members of the P2Y12-R subfamily of P2Y receptors. However, most studies of P2Y14-R-promoted signaling have relied on overexpression of either Gα16 or a chimera of Gαq (Gαq/i) that engineers coupling of this Gi-coupled receptor to activation of phospholipase C (Chambers et al., 2000; Freeman et al., 2001; Fricks et al., 2008). Using C6 rat glioma cells stably expressing the human P2Y14-R, we recently illustrated that adenylyl cyclase activity in both intact cells and isolated membranes was inhibited by UDP-glucose in a concentration- and pertussis toxin-sensitive manner (Fricks et al., 2009). Based on these and similar results obtained with hP2Y14-HEK293 cells, we reasoned that cells displaying P2Y14-R-promoted inhibition of adenylyl cyclase provide a more physiologically relevant system for quantification of P2Y14-R-dependent signaling over an engineered signaling system that depends on overexpression of a G protein that activates phospholipase C. Given our recent observation of activities of UDP at the P2Y14-R transiently expressed with an unnatural chimeric G protein, Gαq/i, in COS-7 cells, we believed it important to quantify the relative actions of UDP and UDP-glucose for promotion of a native cell signaling response in cell lines stably expressing the human P2Y14-R.
Whereas UDP-glucose had no effect on cyclic AMP accumulation in wild-type HEK293 cells (Fig. 1A), incubation of P2Y14-HEK293 cells with this nucleotide sugar resulted in a concentration-dependent inhibition of forskolin-stimulated cyclic AMP accumulation (Fig. 1B). Likewise, UDP had no effect in wild-type HEK293 cells (Fig. 1A) but promoted a concentration-dependent decrease in cyclic AMP accumulation in P2Y14-HEK293 cells. The potency (Table 1) and maximal effect (Table 2) of UDP for inhibition of forskolin-stimulated cyclic AMP accumulation were very similar to the potency and maximal effect of UDP-glucose. As was previously observed with UDP-glucose (Fricks et al., 2009), UDP had no effect on cyclic AMP accumulation in P2Y14-HEK293 cells in which GPCR-promoted activation of Gi was inhibited by preincubation of cells with pertussis toxin (Fig. 1C).
The effect of UDP on cyclic AMP accumulation was observed in several different cell lines cloned from hP2Y14-R virus-infected HEK293 cells (data not shown). Although these results strongly suggest that UDP is a robust natural agonist of this receptor, we concluded it prudent to test possible agonist action of UDP at P2Y14-R stably expressed in other cell backgrounds. Therefore, activity of UDP was assessed in a stable cell line (P2Y14-C6 cells) that we recently developed (Fricks et al., 2009), as well as in a third stable cell line (P2Y14-CHO cells) that was generated as described under Materials and Methods. Again, neither UDP-glucose nor UDP affected basal or forskolin-stimulated cyclic AMP accumulation in wild-type C6 (Fig. 2A) or CHO (data not shown) cells. In contrast, concentration-dependent effects of both UDP-glucose and UDP were observed in P2Y14-C6 (Fig. 2B) and P2Y14-CHO (Fig. 2C) cells. The maximal inhibition of forskolin-stimulated cyclic AMP accumulation observed with UDP was similar to that observed with UDP-glucose in these two cell lines (Table 2), and the EC50 values of UDP and UDP-glucose also were similar (Table 1). The effect of both UDP-glucose and UDP on cyclic AMP accumulation was blocked by preincubation of P2Y14-C6 cells (Fig. 3A) or P2Y14-CHO cells (Fig. 3B) with pertussis toxin.
The agonist activity of other naturally occurring nucleoside diphosphates was tested in P2Y14-HEK293 cells. Both CDP and GDP exhibited agonist activities, although the potencies observed were more than 100-fold less than that observed with UDP (Fig. 4). The effect of ADP, which is the cognate agonist of the P2Y1, P2Y12, and P2Y13 receptors, was also examined. Incubation of P2Y14-HEK293 cells with low micromolar concentrations of ADP resulted in a reproducible increase in cyclic AMP accumulation, and high micromolar concentrations of ADP reduced cyclic AMP accumulation to levels similar to that observed with forskolin alone. The stimulatory effect of ADP also was observed in wild-type HEK293 cells, but the small inhibitory effect observed at high concentrations of ADP was not (data not shown). Thus, ADP exhibited only P2Y14-R-dependent activity at very high concentrations relative to those necessary for agonist effects of UDP. A potential explanation of the stimulatory effect of ADP in both wild-type and P2Y14-HEK293 cells is that ectoenzymes convert ADP to adenosine, which in turn activates an endogenous A2B adenosine receptor known to be present on HEK293 cells (Cooper et al., 1997; Mundell et al., 1999).
Potential agonist activities of the UDP analogs, UDPβS (Malmsjö et al., 2000) and 2-thio-UDP (Besada et al., 2006) also were examined in P2Y14-HEK293 cells. As is illustrated in Fig. 5, both UDPβS (EC50 = 26 ± 11 nM, n = 3) and 2-thio-UDP (EC50 = 2 ± 1 nM, n = 3) were potent agonists at the hP2Y14-R.
We recently reported that stable expression of the human P2Y14 receptor in HEK293 cells confers a robust MAP kinase signaling response to UDP-glucose (Fricks et al., 2009). Therefore, the capacity of UDP to promote P2Y14-R-dependent phosphorylation of ERK1/2 also was examined. As illustrated in Fig. 6, 10 μM UDP had no effect on the MAP kinase response in wild-type HEK293 cells but promoted marked ERK1/2 phosphorylation in P2Y14-HEK293 cells. Thus, as was observed in measurements of cyclic AMP accumulation, quantification of MAP kinase signaling also reveals that UDP and UDP-glucose exhibit similar agonist activities at the P2Y14 receptor.
The results presented thus far with the human P2Y14-R stably expressed in three different cell types strongly suggest that UDP is a potent agonist at this receptor. We also recently discovered that the P2Y14-R is natively expressed in HL-60 promyeloleukemia cells (Fricks et al., 2009). Whereas neither P2Y14-R mRNA nor a MAP kinase signaling response to UDP-glucose was detectable in wild-type HL-60 cells, differentiation of these cells by addition of DMSO to the growth medium resulted in expression of P2Y14-R mRNA as well as phosphorylation of ERK1/2 in response to UDP-glucose. Thus, HL-60 cells also were studied to examine whether UDP activates signaling responses downstream of a natively expressed P2Y14-R. ERK1/2 phosphorylation was observed in response to UDP in differentiated HL60 cells (data not shown), but further experiments revealed that a robust response to UDP also was seen in wild-type HL60 cells in the absence of P2Y14-R expression (Fig. 7A). Reverse transcription-polymerase chain reaction analyses revealed that the response observed to UDP in wild-type HL60 cells is probably due to the presence of a UDP-activated P2Y6-R, because mRNA for this receptor is prominently expressed in both wild-type and differentiated HL60 cells (data not shown). Although quantification of inhibition of cyclic AMP accumulation potentially might allow resolution of the effects of UDP on the Gi-coupled P2Y14-R versus Gq-coupled P2Y6-R in these cells, we have been unable to observe inhibition of adenylyl cyclase in response to UDP-glucose or formyl-Met-Leu-Phe, which activates a Gi-linked receptor for chemotactic peptides in HL-60 cells (Fricks et al., 2009).
In the absence of availability of another cell line that natively expresses the P2Y14-R, we considered a pharmacological approach to show that the P2Y14-R natively expressed on differentiated HL-60 cells responds to agonists in addition to nucleotide sugars. As illustrated above (Fig. 5), two UDP analogs activated the P2Y14-R stably expressed in HEK293 cells. Because these two molecules also are potent activators of the P2Y6-R (data not shown), they are not useful for selectively activating the P2Y14-R of HL-60 cells. However, our recent chemical syntheses (A. Das, H. Ko, M. O. Barrett, L. B. Burianek, T. K. Harden, and K. A. Jacobson, submitted) have identified analogs of UDP that activate the P2Y14-R without activating the P2Y6-R, and the relative activities of two of these, MRS2802 and MRS2907, at the P2Y14-R versus P2Y6-R are illustrated in Fig. 7. MRS2802 inhibited forskolin-stimulated cyclic AMP accumulation in P2Y14-HEK293 cells with an EC50 of approximately 50 nM (Fig. 7B, left), whereas little inositol phosphate response to this molecule was observed at concentrations up to 10 μM in 1321N1 human astrocytoma cells stably expressing the human P2Y6-R (Fig. 7B, right). MRS2907 also selectively activated the P2Y14-R over the P2Y6-R (Fig. 7C). Given the P2Y14-R selectivity of these two UDP analogs, we examined their capacity to stimulate ERK1/2 phosphorylation in wild-type and differentiated HL-60 cells. Neither MRS2802 nor MRS2907 promoted ERK1/2 phosphorylation in wild-type HL-60 cells (Fig. 7D), but as was the case with UDP-glucose, a MAP kinase signaling response to both analogs was observed after induction of expression of the P2Y14-R by differentiation of these cells by treatment with DMSO. These results are consistent with the idea that UDP analogs, and by deduction UDP itself, are potent agonists of the P2Y14-R in a native environment.
Discussion
The results of this study illustrate that UDP is a cognate agonist of the human P2Y14-R. Agonist activity of UDP was observed in three different cell backgrounds in which the capacity of the human P2Y14-R to promote inhibition of forskolin-stimulated adenylyl cyclase activity was quantified. Given that the potency of UDP is similar to that of UDP-glucose and other nucleotide sugars for activation, we conclude that UDP is a physiologically important agonist of the human P2Y14-R.
Previous studies of P2Y14-R have focused almost entirely on the activity of uridine nucleotide sugars. Chambers and colleagues (Chambers et al., 2000; Freeman et al., 2001) first reported that the orphan GPCR, KIAA0001, subsequently named the P2Y14-R, is activated by UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine. In contrast, a broad range of other nucleotide sugars and nucleotides, including UDP, were reported to be inactive at this receptor. These initial pharmacological studies used three different systems to quantify agonist activities. A yeast transcription reporter assay system was applied that tested human P2Y14-R-dependent activity during overnight incubation with potential agonists. The human P2Y14-R also was transiently expressed in HEK293 cells with Gα16, a heterotrimeric G protein that promiscuously couples to most agonist-activated GPCR and confers agonist-dependent activation of phospholipase C and mobilization of Ca2+ to these receptors. Finally, membranes were prepared from HEK293 cells that stably expressed the human P2Y14-R, and UDP-glucose-promoted binding of [35S]GTPγS to endogenous G proteins was quantified.
It is unclear why agonist activity of UDP was not observed in early studies of the P2Y14-R. The yeast transcription assay and assays that take advantage of promiscuous coupling of GPCR to Gα16 provide robust assessments of GPCR activity. However, because neither of these systems measure coupling to the cognate heterotrimeric G protein known to mediate P2Y14-R-promoted signaling responses, it is possible that a receptor conformation is favored in these test systems that does not fully reflect the ligand binding selectivities and agonist activities of the physiological receptor coupling to its cognate heterotrimeric G protein and signaling pathway. On the other hand, these early studies included assays of agonist-promoted binding of [35S]GTPγS to endogenous G proteins in membranes from P2Y14-R-expressing HEK293 cells, and UDP-glucose promoted increases in [35S]GTPγS binding that were 40% greater than basal. This activity apparently reflected agonist-induced coupling to a Gα-subunit(s) of the Gi family because it was inhibited by pertussis toxin. It is noteworthy that concentration effect curves for four different nucleotides, including UDP, were generated with this test system, and that in contradistinction to the stimulatory effect of UDP-glucose, all four nucleotides inhibited [35S]GTPγS binding in a concentration dependent manner. However, this effect was not dependent on expression of the P2Y14-R, was not blocked by pertussis toxin, and therefore apparently represented a nonspecific effect on binding of [35S]GTPγS to unspecified proteins. Given the magnitude of this inhibitory effect of nucleotides and the relatively small magnitude of P2Y14-R-dependent UDP-glucose-promoted increases in [35S]GTPγS binding, the sensitivity of this test system apparently was not sufficient to reveal an agonist action of UDP at the P2Y14-R.
We recently reported that UDP exhibits competitive antagonist/partial agonist activity in measurements of [3H]inositol phosphate accumulation in COS-7 cells transiently coexpressing the hP2Y14-R with a chimeric G protein Gαq/i that confers a phospholipase C/Ca2+ signaling response to Gi-activating receptors (Fricks et al., 2008). It is noteworthy that UDP seemed to be a full agonist in parallel studies with the rat P2Y14-R. Moreover, our unpublished studies examining activities of the human P2Y14-R cotransfected with Gαq/i in COS-7 cells show that an antagonist/partial agonist action of UDP is observed only at very low levels of receptor expression. With higher receptor expression, UDP exhibits maximal agonist effects that are similar to those of UDP-glucose. As we speculated above concerning studies with Gα16, a conformation of the P2Y14-R may be favored when this receptor is coupled to Gαq/i, which results in ligand binding selectivities and agonist activities that are not altogether consistent with activities of the receptor obtained when coupled to its cognate heterotrimeric G protein. The idea that relative receptor activation by ligands is influenced by both the levels of signaling proteins and the particular G protein signaling system studied is supported by previous studies of a variety of G protein-coupled receptors (Cordeaux et al., 2004; Kenakin, 2007; Urban et al., 2007). The work of Ault and Broach using directed evolution in yeast first suggested an interaction of UDP with the hP2Y14-R, and their studies with various mutants of the human P2Y14-R also suggested a range of activities of this diphosphate relative to activities observed with UDP-glucose (Ault and Broach, 2006).
Our data with P2Y14-HEK293, P2Y14-C6, and P2Y14-CHO cells all reveal potent agonist activity of UDP. The maximal inhibitory effect on adenylyl cyclase observed with UDP was the same as that of UDP-glucose, which is consistent with the idea that UDP is a full agonist at the hP2Y14-R. However, we cannot unequivocally rule out the possibility that under limiting receptor concentrations, UDP would prove to be a partial agonist. A radioligand is not available for quantification of P2Y14-R; consequently, the density of receptors cannot be determined in these cells with current technology. Nonetheless, viral expression of GPCR predictably results in receptor levels that do not substantively exceed that of native receptors. We also have isolated a series of clonal P2Y14-HEK293 cell lines that exhibit varying amounts of expression of P2Y14-R quantified by surface epitope-tagging. UDP and UDP-glucose are similarly efficacious in each of these cell lines tested. Finally, the presence of an endogenous UDP-activated P2Y6-R on HL-60 promyeloleukemia cells precludes straightforward testing of UDP as a P2Y14-R agonist on these cells. However, the fact that P2Y14-R-selective analogs of UDP promoted phosphorylation of ERK1/2 in differentiated, but not in undifferentiated, HL-60 cells also is consistent with the conclusion that UDP is an agonist of the P2Y14-R.
Although the P2Y14-R has been thought to be solely regulated by UDP sugars, we conclude that this signaling protein is also significantly activated by UDP. Lazarowski and colleagues have hypothesized that P2Y14-R-activating nucleotide sugars arise from cellular protein secretory pathways (Lazarowski et al., 2003). Although we have illustrated that mechanical stimulation promotes release of UTP into the extracellular medium from a broad range of cells (Lazarowski et al., 1997; Lazarowski and Harden, 1999), the source of extracellular UDP remains unclear. One idea is that ecto-nucleotide triphosphate-diphosphohydrolase 2, which converts nucleoside triphosphates to nucleoside diphosphates (Zimmermann, 2000), converts UTP to UDP at the cell surface. For example, the pharmacological effects of ATP at the ADP-activated P2Y1 receptor are markedly augmented by expression of ectoNTPDase2 (Alvarado-Castillo et al., 2005). Alternatively, UDP could be released from a secretory pathway, as suggested by Tatur et al. (2008). Because UDP and UDP-sugars participate in glycosylation reactions in the Golgi lumen, one speculation is that both species are released concomitantly during vesicle exocytosis. Perhaps a common source of the two agonists in part explains the evolution of a receptor that is dually activated by UDP and UDP sugars. The P2Y14-R has been implicated in regulation of immune responses (Fumagalli et al., 2003; Skelton et al., 2003; Müller et al., 2005; Scrivens and Dickenson, 2005; Scrivens and Dickenson, 2006), and it will be important to establish that both UDP-glucose and UDP play important and possibly differing roles in this function.
An intriguing aspect of the discovery that UDP is an agonist of the P2Y14-R is that the P2Y14-R and the UDP-activated P2Y6-R have overlapping cellular distributions (Burnstock and Knight, 2004). One of the best understood physiological responses mediated by P2Y-R is the ADP-promoted aggregation of platelets, which requires coordinated activation of both the Gi-coupled P2Y12 receptor and the Gq-coupled P2Y1 receptor (Gachet, 2006). We hypothesize that certain physiological responses to extracellular UDP also require coordinated activation of the Gq-activating P2Y6-R and the Gi-activating P2Y14-R. A current goal of our research is to develop receptor selective ligands that allow pharmacological resolution of the activities of these two receptors in native tissues.
Footnotes
This work was supported by the National Institutes of Health National Institute of General Medicine [Grant GM38213]; the National Institutes of Health National Heart, Lung, and Blood Institute [Grant HL34322]; and by the Intramural Research Program of the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.058578
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ABBREVIATIONS:
- P2Y14-R
- P2Y14 receptor
- P2Y6-R
- P2Y6 receptor
- hP2Y14-R
- human P2Y14 receptor
- P2Y14-HEK293 cells
- human embryonic kidney 293 cells stably expressing the human P2Y14 receptor
- P2Y14-C6 cells
- C6 rat glioma cells stably expressing the human P2Y14 receptor
- P2Y14-CHO cells
- Chinese hamster ovary cells stably expressing the human P2Y14 receptor
- DMEM
- Dulbecco's modified Eagle's medium
- DMSO
- dimethyl sulfoxide
- ERK
- extracellular signal-regulated kinase
- UDPβS
- uridine 5′-O-thiodiphosphate
- MAP
- mitogen-activated protein
- MRS2802
- α,β-difluoromethylene-UDP
- MRS2907
- 2-thio-UDP-β-propyl ester
- GTPγS
- guanosine 5′-O-(3-thio)triphosphate
- GPCR
- G protein-coupled receptor.
- Received June 12, 2009.
- Accepted September 16, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics