QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.108.136309v1 325/2/588    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fricks, I. P. Articles by Harden, T. K. Search for Related Content PubMed PubMed Citation Articles by Fricks, I. P. Articles by Harden, T. K. Pubmed/NCBI databases Compound via MeSH Substance via MeSH

CELLULAR AND MOLECULAR

UDP Is a Competitive Antagonist at the Human P2Y₁₄ Receptor

Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (I.P.F., S.M., R.C., E.R.L., R.A.N., T.K.H.); and Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (K.A.J.)

Received January 7, 2008; accepted January 29, 2008.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
G protein-coupled P2Y receptors (P2Y-R) are activated by adenine and uracil nucleotides. The P2Y₁₄ receptor (P2Y₁₄-R) is activated by at least four naturally occurring UDP sugars, with UDP-glucose (UDP-Glc) being the most potent agonist. With the goal of identifying a competitive antagonist for the P2Y₁₄-R, UDP was examined for antagonist activity in COS-7 cells transiently expressing the human P2Y₁₄-R and a chimeric G protein that couples Gi-coupled receptors to stimulation of phosphoinositide hydrolysis. UDP antagonized the agonist action of UDP-Glc, and Schild analysis confirmed that the antagonism was competitive (pK_B = 7.28). Uridine 5'-O-thiodiphosphate also antagonized the human P2Y₁₄-R (hP2Y₁₄-R) with an apparent affinity similar to that of UDP. In contrast, no antagonist activity was observed with ADP, CDP, or GDP, and other uracil analogs also failed to exhibit antagonist activity. The antagonist activity of UDP was not observed at other hP2Y-R. In contrast to its antagonist action at the hP2Y₁₄-R, UDP was a potent agonist (EC₅₀ = 0.35 µM) at the rat P2Y₁₄-R. These results identify the first competitive antagonist of the P2Y₁₄-R and demonstrate pharmacological differences between receptor orthologs.

The human P2Y₁₄-R (hP2Y₁₄-R) was identified as the eighth legitimate member of the P2Y receptor family (Chambers et al., 2000). UDP-glucose (UDP-Glc) was proposed to be the endogenous agonist for the P2Y₁₄ receptor (P2Y₁₄-R), with UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine acting as less potent P2Y₁₄-R agonists. P2Y₁₄-R mRNA was detected in a broad range of human tissues including placenta, stomach, intestine, adipose, brain, lung, spleen, and heart, and it was also found in specialized cells such as circulating neutrophils (Chambers et al., 2000; Scrivens and Dickenson, 2006). The rat and mouse P2Y₁₄-R exhibit 80 and 83% amino acid identity to the human receptor, and both rodent orthologs are activated by known agonists with a similar profile to that of the hP2Y₁₄-R (Freeman et al., 2001).

Cellular UDP-Glc is released in a constitutive manner into the medium of a broad range of cell types. Although most extracellular nucleotides are metabolized quickly, UDP-Glc accumulates in the medium of several cell lines (Lazarowski et al., 2003). The mechanisms of UDP-Glc release and extracellular metabolism remain unclear. Observations of UDP-Glc-promoted signaling were reported in multiple types of immune cells (Fumagalli et al., 2003; Skelton et al., 2003; Müller et al., 2005; Scrivens and Dickenson, 2005), suggesting that the P2Y₁₄-R may have a yet-to-be-defined role in the regulation of immune system homeostasis.

Characterization of the P2Y₁₄-R has been slowed by the lack of a selective competitive antagonist. We have identified and developed novel, selective ligands for several P2Y receptors that have proven useful for pharmacological resolution of molecularly defined P2Y-R in cells and tissues (Boyer et al., 1996; Houston et al., 2006, 2008; Jacobson et al., 2006). Accordingly, we are interested in identifying a selective antagonist for the P2Y₁₄-R.

Ault and Broach (2006) recently used a yeast model system in which various nucleotides and nucleotide sugars were examined for their ability to stimulate growth of mutant P2Y₁₄-R-expressing yeast cells in studies focused on the identification of mutant P2Y₁₄ receptors with differential agonist sensitivities. Studies performed using one of these mutant receptors revealed that UDP antagonized UDP-Glc-promoted receptor activation in a concentration-dependent manner (Ault and Broach, 2006). We hypothesized that UDP acts as a competitive antagonist at the wild-type P2Y₁₄-R and, therefore, used a transfected COS-7 cell system to investigate UDP activity at the human and rat P2Y₁₄-R. In this study, we show that UDP is a selective and competitive antagonist of the hP2Y₁₄-R. Thus, signals emanating from extracellular UDP apparently occur as a consequence of activation of the P2Y₆-R as well as through antagonism of the P2Y₁₄-R. We were surprised to find that UDP is a potent full agonist at the rat P2Y₁₄-R.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. UDP-Glc, UDP, ADP, CDP, GDP, and 2-methyl-thio-ADP were purchased from Sigma-Aldrich (St. Louis, MO). Diuridine 5',5'-triphosphate (UP₃U) was synthesized according to the methods detailed in Pendergast et al. (2001). The source of diuridine 5',5'-tetraphosphate (UP₄U) was as previously reported (Ivanov et al., 2007). ATP and UTP were purchased from GE Healthcare (Piscataway, NJ). [³²P]PPi was synthesized as described previously (Lazarowski et al., 2003). Uridine 5'-O-thiodiphosphate (UDPβS) as well as a mammalian expression vector for ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) were generous gifts from Dr. José Boyer of Inspire Pharmaceuticals (Durham, NC).

Cell Culture and Transfection. COS-7 cells were grown on 12-well culture dishes and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 4 mM L-glutamine at 37°C in a 10% CO₂ environment. FuGENE 6 (Roche Applied Science, Indianapolis, IN) was used as the transfection reagent following the manufacturer's protocol. Cells were transfected 48 h before assay with pcDNA3.1 expression vectors encoding either the human or rat P2Y₁₄ receptor with an N-terminal hemagglutinin epitope tag. The expression vector for the hP2Y₁₄-R was obtained as reported previously (Lazarowski et al., 2003). Transfections also included pcDNA3.1-G_q/i, a vector that directs expression of a chimera of Gq containing the last five amino acids of Gi. This chimeric G protein promotes activation of phospholipase C through Gi-coupled receptors (Coward et al., 1999). The levels of basal inositol phosphates increased markedly in COS-7 cells upon expression of human or rat P2Y₁₄-R and G_q/i. Because we previously illustrated that UDP-sugars are basally released by various cell types (Lazarowski et al., 2003), in some experiments pcDNA3.1-expressing ENPP1 was cotransfected with the goal of hydrolyzing extracellular nucleotide sugars and potentially decreasing inositol phosphate accumulation in the absence of added P2Y₁₄-R agonists.

Inositol Phosphate Accumulation Assay. Cells were labeled 8 to 18 h before assay with 0.5 to 3 µCi/well myo-[³H]inositol (American Radiolabeled Chemicals, St. Louis, MO) in inositol-free and serum-free DMEM. Assays were initiated with the addition of 10 mM LiCl with or without drugs, and incubations continued for 45 min at 37°C. Reactions were stopped by aspiration of medium and addition of ice-cold 50 mM formic acid. After neutralization with 150 mM ammonium hydroxide, [³H]inositol phosphates were isolated by Dowex column chromatography as described previously (Nakahata and Harden, 1987). Stable cell lines for P2Y₁, P2Y₂, P2Y₄, P2Y₆, or P2Y₁₁ receptors were generated in 1321N1 human astrocytoma cells as described previously (Nicholas et al., 1996; Kennedy et al., 2000). Experiments testing the potential activity of UDP at the P2Y₂-R and P2Y₄-R included hexokinase to eliminate contamination of UTP as previously described in Nicholas et al. (1996). In brief, UDP was treated with 10 U/ml hexokinase in the presence of 22 mM glucose for 1 h at 37°C, and 1 U/ml hexokinase was included in the assay buffer for the duration of the incubation. Variability in cpm of [³H]-inositol phosphate accumulation across experiments occurred due to differences in the amount of [³H]inositol used for labeling and/or the duration of the prelabeling period.

Measurement of UDP-Glc in the Cell Medium. Quantification of UDP-Glc was performed as described previously (Lazarowski et al., 2003). In brief, incubations were in a final volume of 150 µl containing known or unknown amounts of UDP-Glc, 25 mM HEPES, pH 7.4, 0.5 U/ml UDP-glucose pyrophosphorylase from baker's yeast (Sigma-Aldrich), and 100 nM [³²P]PPi (200,000 cpm). Incubations were terminated by addition of 0.3 mM PPi and immediate heating of samples for 2 min at 95°C, and formation of [³²P]UTP was quantified by high-performance liquid chromatography as described previously (Lazarowski et al., 2003).

Quantification of P2Y₁₄-R Expression. Cells were seeded in 12-well plates at 5 x 10⁴ cells/well 3 days before assay and transfected with mammalian expression vectors as described above. Cells were fixed with 0.4 ml of 4% paraformaldehyde for 30 min at room temperature, washed twice with 1 ml of Hanks' balanced salt solution plus Ca²⁺/Mg²⁺, and incubated for 30 min at room temperature with 0.4 ml of DMEM plus 50 mM HEPES, pH 7.1, and 10% fetal bovine serum. Cells were incubated with mouse HA.11 monoclonal antibody at a 1:1000 dilution in 0.4 ml of medium for 1 h at room temperature. After two washes with 1 ml of Hanks' balanced salt solution plus Ca²⁺/Mg²⁺, cells were incubated with [¹²⁵I]rabbit anti-mouse IgG antibody diluted to 1:500 in 0.4 ml of medium for 2 h at room temperature. After another series of washing steps, cells were solubilized in 0.4 ml of 1 M NaOH overnight and transferred to glass tubes for quantification of radioactivity in a gamma counter.

Rat P2Y₁₄-R Subcloning. The rat homolog of the P2Y₁₄-R (rP2Y₁₄-R) was amplified from rat genomic DNA using Pfu polymerase (Stratagene, Carlsbad, CA) with the following primers: (5'-GAGACGCGTCCGACAACACAACAACCACAGAAC-3') and (5'-AGACTCGAGTTACAAAGTATCTGTGCTTTCC-3'). The primers contained either a MluI (upstream primer) or a XhoI (downstream primer) restriction site, respectively (sites are underlined), to facilitate cloning. The amplification conditions were as follows: 94°C for 5 min; 35 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 90 s; and a final extension for 4 min at 72°C. The amplified rP2Y₁₄-R fragment was digested with MluI and XhoI, purified, and ligated into a similarly digested, modified pcDNA3 expression vector, which fuses a hemagglutinin epitope to Asp-2 at the N terminus of the receptor. An individual clone encoding the receptor was sequenced and found to be identical to the published sequence (Freeman et al., 2001).

Data Analyses. The results from each experiment are expressed as the mean ± S.E. from triplicate samples and were analyzed using Prism 4.0 software (GraphPad Software Inc., San Diego, CA). All experiments were repeated at least three times with similar results. Schild analysis was performed using EC₅₀ values from the concentration-effect curves for UDP-Glc generated in the absence and presence of increasing concentrations of UDP. The pK_B was calculated using the equation: log ([A']/[A]–1) = log [B]–log pK_B (Arunlakshana and Schild, 1959), where [A'] is the concentration of UDP-Glc necessary to produce 50% of the maximal effect in the presence of antagonist [B], and [A] is the concentration of agonist necessary to produce 50% of the maximal effect in the absence of antagonist. Drug response data presented in Figs. 3A, 5, A–D, and 7B are normalized as a percentage of the response observed with a maximally effective concentration (usually 10 µM) of UDP-Glc. Statistics were carried out using the Student's t test.

View larger version (24K): [in this window] [in a new window]   Fig. 3. UDP is a competitive antagonist at the hP2Y14-R. A, [3H]inositol-labeled COS-7 cells coexpressing hP2Y14-R and Gq/i were incubated with LiCl (10 mM) and increasing concentrations of UDP-glucose in the absence or presence of the indicated concentrations of UDP: , buffer; , 0.1 µM; , 0.3 µM; , 1 µM; , 3 µM; , 30 µM; and , 100 µM. Data shown are the means ± S.E. calculated from triplicate samples and are representative of the results obtained in three independent experiments. B, EC50 values from the concentration-effect curves in A were used for Schild regression analysis. The data shown are results from a representative experiment repeated three times to yield a mean pKB of 7.28 ± 0.04 and a slope of 1.15 ± 0.06.

View larger version (32K): [in this window] [in a new window]   Fig. 5. UDP is unique among nucleoside diphosphates for its antagonist effect at the hP2Y14-R. CDP (A), ADP (B), GDP (C), or UDP (D) (10 or 100 µM) and LiCl (10 mM) were applied simultaneously with 3 µM UDP-Glc to [3H]inositol-labeled COS-7 cells transiently expressing hP2Y14-R and Gq/i. Data were normalized to values from maximal activation of hP2Y14-R by UDP-Glc alone, and the LiCl alone value was subtracted from each data point. The data shown in A, C, and D are results from a representative experiment repeated three times. The data shown in B are the average of results from four experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Agonist effect of UDP-Glc and UDP at the rP2Y14-R. A, COS-7 cells transfected with an expression vector for Gq/i and with empty vector or rP2Y14-R were [3H]inositol-labeled and incubated with 10 mM LiCl in the absence or presence of 10 µM UDP-Glc. B, [3H]inositol-labeled COS-7 cells transiently expressing the rP2Y14-R and Gq/i were incubated with 10 mM LiCl and UDP-Glc or UDP at the indicated concentrations, and [3H]inositol phosphate accumulation was quantified as described under Materials and Methods. The data shown are the means ± S.E. calculated from triplicate samples and are results from a representative experiment repeated three times, yielding an EC50 of 0.28 ± 0.05 µM for UDP-Glc and 0.35 ± 0.17 µM for UDP. Data were normalized to the maximal activation of rP2Y14-R by UDP-Glc. C, cells incubated with UDP-Glc (10 µM) + UDP (100 µM) exhibited no difference in [3H]inositol phosphate accumulation compared to accumulation in the presence of either agonist alone. The data shown are the means ± S.E. calculated from triplicate samples and are representative of results obtained in three independent experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 1. hP2Y14-R- and Gq/i-dependent increases in [3H]inositol phosphate accumulation. COS-7 cells were transfected with empty vector or expression vectors for hP2Y14-R and/or Gq/i as described under Materials and Methods. Cells were labeled with [3H]inositol for 18 h before assay. LiCl (10 mM) buffer was added to the cells to inhibit inositol monophosphatase, and the cells were simultaneously incubated in the absence or presence of 10 µM UDP-Glc for 45 min. [3H]Inositol phosphates were isolated as described under Materials and Methods. The data shown are the means ± S.E. calculated from triplicate samples and are representative of the results obtained in three independent experiments.

	Results

Top Abstract Materials and Methods Results Discussion References

View larger version (21K): [in this window] [in a new window]   Fig. 2. Coexpression of ENPP1 reduces the basal activation of the hP2Y14-R. COS-7 cells were cotransfected with expression vectors for hP2Y14-R and Gq/i with (+) or without (–) an expression vector for ENPP1. A, [3H]inositol-labeled cells were incubated with 10 mM LiCl in the absence or presence of UDP-Glc. *, p < 0.01. B, [3H]inositol-labeled cells were incubated with 10 mM LiCl in the absence or presence of UDP-Glc at the indicated concentrations. The data shown are the means ± S.E. calculated from triplicate determinations.

Antagonist Effect of UDP at the hP2Y₁₄-R. Four UDP sugars were identified as agonists at the hP2Y₁₄-R, and neither UTP nor UDP exhibited agonist activity (Chambers et al., 2000). To determine whether UDP is an antagonist at the wild-type hP2Y₁₄-R, we generated a series of concentration-effect curves for UDP-Glc-promoted stimulation of phospholipase C in the presence of increasing concentrations of UDP (Fig. 3A). UDP caused a parallel rightward shift of the UDP-Glc concentration-effect curve, and Schild analysis (Fig. 3B) confirmed that the antagonism produced by UDP was competitive (slope = 1.15 ± 0.06, n = 3). The pK_B of UDP for antagonism of the hP2Y₁₄-R was 7.28 ± 0.04.

We also assessed whether UDP exhibited antagonist activity at other P2Y-R stably expressed in 1321N1 human astrocytoma cells. P2Y₁-R was maximally activated by 1 µM 2MeSADP (Fig. 4A), P2Y₂-R and P2Y₄-R were each activated by 3 µM UTP (Fig. 4, B and C), and P2Y₁₁-R was activated by 100 µM ATP (Fig. 4D). Although 10 µM UDP completely blocked a near-maximal concentration of UDP-Glc at the hP2Y₁₄-R (Fig. 3), UDP had no effect at any other P2Y receptors tested. Thus, we conclude that UDP is a selective antagonist at the hP2Y₁₄-R.

View larger version (31K): [in this window] [in a new window]   Fig. 4. UDP is a selective antagonist at the hP2Y14-R. [3H]Inositol-labeled 1321N1 human astrocytoma cells stably expressing either the human P2Y1-R (A), human P2Y2-R (B), human P2Y4-R (C), or human P2Y11-R (D) were incubated for 30 min with 10 mM LiCl with the cognate agonist (indicated), or 10 µM UDP, or both agonist and UDP, and inositol phosphate accumulation was quantified as described under Materials and Methods. The data shown are the means ± S.E. calculated from triplicate samples and are representative of the results obtained in three or more independent experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Antagonist effect of UDPβS at the hP2Y14 -R. [3H]Inositol-labeled COS-7 cells transiently expressing hP2Y14-R and Gq/i were incubated with 10 mM LiCl in the absence () or presence () of 1 µM UDP-Glc in the presence of the indicated concentrations of UDPβS for 30 min. The data shown are the means ± S.E. of triplicate determinations, and the results are representative of those obtained in three experiments.

Effects of UDP at the rP2Y₁₄-R. Because pharmacological studies are often carried out with rat or mouse tissues, it is important to assess whether receptor orthologs exhibit pharmacological selectivities similar to those of human P2Y-R. Based on the precedent that ATP acts as an antagonist at the human P2Y₄-R but is an agonist at the rat P2Y₄-R (Bogdanov et al., 1998; Kennedy et al., 2000), we compared the action of UDP at the rP2Y₁₄-R with its action at the hP2Y₁₄-R. The rat P2Y₁₄-R, which exhibits approximately 80% amino acid sequence identity to the hP2Y₁₄-R and almost 90% identity in the transmembrane regions alone, was reported to display a similar UDP-sugar selectivity to that of the hP2Y₁₄-R (Freeman et al., 2001), but the actions of other uridine nucleotides on the rP2Y₁₄-R have not been reported.

Expression of either the rP2Y₁₄-R or G_q/i in COS-7 cells had no effect on [³H]inositol phosphate accumulation compared with untransfected cells, but coexpression of receptor and G_q/i resulted in markedly increased basal accumulation (Fig. 7A). Consistent with other reports (Freeman et al., 2001), UDP-Glc was a potent agonist at the rP2Y₁₄-R (Fig. 7B). Whereas UDP had no effect on inositol phosphate accumulation in wild-type COS-7 cells, in cells expressing G_q/i alone, or in cells expressing the rP2Y₁₄-R alone (data not shown), concentration of UDP-dependent increases in formation of [³H]inositol phosphates occurred in COS-7 cells coexpressing the rat P2Y₁₄-R with G_q/i (Fig. 7B). The maximal stimulatory effect observed with UDP was similar to that observed with UDP-Glc, as were the EC₅₀ values of UDP (0.35 ± 0.17 µM) and UDP-Glc (EC₅₀ = 0.28 ± µM). No additivity was observed between UDP and UDP-Glc on rP2Y₁₄ -R-promoted [³H]inositol phosphate formation (Fig. 7C).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, we show that UDP is a competitive antagonist at the human P2Y₁₄-R, and this action is receptor-selective because UDP does not inhibit agonist-promoted activation of other hP2Y receptors. Moreover, the activity of UDP at the P2Y₁₄-R is species-dependent because we observed that UDP is a potent, and apparently full, agonist at the rat P2Y₁₄-R.

Chambers et al. (2000) reported in their initial study of the hP2Y₁₄-R that UDP has no agonist activity, and we observed similar results in the studies reported here. Using a reporter system in yeast, Ault and Broach (2006) generated a mutant hP2Y₁₄-R displaying a mutation in intracellular loop 1 and various mutations in several of the transmembrane regions. This mutant, selected for its ability to support growth of yeast at lower concentrations of UDP-Glc than the wild-type receptor, exhibited an enhanced UDP-Glc-stimulated response that was inhibited by UDP, and a K_B in the micromolar range was reported. The >20-fold higher potency of UDP observed in our studies probably reflects large differences in the assay systems used. For example, whereas incubations with nucleotide were continued for minutes in the current study, they were continued for hours in assays measuring P2Y₁₄-R-mediated regulation of growth of yeast. Our results illustrate that UDP is a potent, competitive antagonist of the wild-type hP2Y₁₄-R.

Demonstration of antagonist action at the hP2Y₁₄-R suggests that UDP may have broader physiological importance as an extracellular signaling molecule than has been previously appreciated. Both UDP and UDP-glucose are known to be released from cells, although the mechanisms of their release remain unclear. UDP is the most potent and selective agonist of the hP2Y₆-R (Lazarowski and Harden, 1994; Communi et al., 1996), and physiological responses attributed to UDP-initiated P2Y₆-R-promoted signaling include modulation of interleukin-8 production in monocytes (Warny et al., 2001) and human mature dendritic cells (Idzko et al., 2004). In addition, UDP was observed to induce a positive inotropic effect in mouse cardiomyocytes (Wihlborg et al., 2006) and to promote ion transport in human placental cytotrophoblast cells (Roberts et al., 2006). Our data indicate that potential contributions of the P2Y₁₄-R to responses associated with UDP must be considered. Whereas reported distribution of P2Y₆-R mRNA overlaps with that of P2Y₁₄-R mRNA in many cells and tissues, such as lung, heart, placenta, and neutrophils (Communi et al., 1996; Moore et al., 2001), it remains unclear whether the two receptor types are coexpressed in the same cells or in different cells that share the same extracellular space.

The actions of extracellular neurotransmitters and hormones are highly regulated by their release, metabolism, and reuptake. The possibility that direct antagonism of GPCR activation occurs by extracellular signaling molecules has been suggested by observations that ATP is a competitive antagonist of the hP2Y₁₂-R (Cusack and Hourani, 1982; Bodor et al., 2003) and of the hP2Y₄-R (Bogdanov et al., 1998; Kennedy et al., 2000). The physiological relevance of UDP antagonism at the hP2Y₁₄-R will be important to investigate, as will the idea that UDP simultaneously activates the hP2Y₆-R while inhibiting the hP2Y₁₄-R.

Our finding that UDP has agonist activity at the rP2Y₁₄-R was surprising. Study of the rat and human receptors under identical conditions rules out trivial explanations for this observation. These data do not unambiguously rule out the possibility that UDP is a partial agonist at the human receptor under some conditions. However, the agonist versus antagonist action of the nucleotide at the rat versus human P2Y₁₄-R has been observed over a broad range of expression levels of these two receptors. Our activity data suggest that the binding affinity of UDP for the human and rat receptors are in fact quite similar, but development of a radioligand binding assay will be necessary to fully assess this assertion. The fact that the maximal agonist activity of UDP was similar to that of UDP-Glc over a broad range of P2Y₁₄-R expression levels (data not shown) suggests that the intrinsic efficacy of UDP at the rat P2Y₁₄-R is similar to that of UDP-Glc.

The differential activity of UDP observed between the rat and human orthologs of the P2Y₁₄-R shares similarities to the actions of ATP at the P2Y₄-R. Kennedy et al. (2000) compared the ligand selectivities of the rat and human P2Y₄ receptors under conditions that minimize the effects of released nucleotides and of extracellular bioconversion of nucleotides, and they observed that ATP is an agonist at the rat P2Y₄-R and a competitive antagonist at the human P2Y₄-R. Residues in the second extracellular loop of the P2Y₄ receptor are the primary determinants for the agonist versus antagonist activity of ATP between the two species orthologs (Herold et al., 2004). Like the P2Y₄-R, the P2Y₁₄-R shares approximately 80% amino acid sequence identity between the rat and human orthologs, with 90% identity when analysis is restricted to transmembrane regions only. Recent work by Ko et al. (2007) defines the human P2Y₁₄ receptor through structure-activity studies in conjunction with molecular modeling studies. Residues in the second extracellular loop of the hP2Y₁₄-R are predicted to interact with the diphosphate moiety and hydroxyl groups on the hexose ring of UDP-Glc. Comparative modeling of the rat P2Y₁₄-R and receptor mutagenesis directed from these predictions may identify key domains responsible for agonist efficacy at the hP2Y₁₄-R.

The mouse P2Y₁₄-R has been cloned and is reported to be activated by the same UDP-sugar agonists as the human and rat receptors (Freeman et al., 2001). The rat and mouse P2Y₁₄-R share 89% overall amino acid sequence identity and are essentially identical in their transmembrane-spanning domains and the second extracellular loop. Thus, we anticipate that the agonist action of UDP observed with the rat receptor will be similarly observed at the mouse P2Y₁₄-R.

The finding that UDP acts as a competitive antagonist at the hP2Y₁₄-R provides an excellent template for rational synthesis of antagonist analogs that exhibit high affinity at the hP2Y₁₄-R. Our structure-activity studies of UDP analogs at the hP2Y₆-R (Besada et al., 2006) also provide potential avenues for development of P2Y₁₄-R antagonists that do not act as ligands for the P2Y₆-R. Synthesis of a hydrolysis-resistant competitive antagonist for the P2Y₁₄-R is an obvious goal, as is a high-affinity radiolabeled antagonist.

UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine were previously identified as full or partial agonists at the human and rodent P2Y₁₄-R (Chambers et al., 2000; Freeman et al., 2001). The pharmacological selectivity for the P2Y₁₄-R is now broadened with the finding that UDP also acts at this receptor. The identification of UDP as a competitive antagonist for the hP2Y₁₄-R provides new insight into the physiological regulation of this receptor, and it should be of pharmacological importance in delineating the functional roles subserved by this signaling protein.

	Acknowledgements

 
We appreciate the excellent technical assistance of Sonia de Castro for the synthesis of UP₃U.

	Footnotes

 
This work was supported by National Institutes of Health Grants GM38213, P01-HL034322, and the National Institutes of Health Intramural Research Program of National Institute of Diabetes and Digestive and Kidney Diseases.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.136309.

ABBREVIATIONS: P2Y-R, P2Y receptor; GPCR, G protein-coupled receptor; hP2Y₁₄-R, human P2Y₁₄-R; UDP-Glc, UDP glucose; P2Y₁₄-R, P2Y₁₄ receptor; UP₃U, diuridine 5',5'-triphosphate; UP₄U, diuridine 5',5'-tetraphosphate; UDPβS, uridine 5'-O-thiodiphosphate; ENPP1, ecto-nucleotide pyrophosphatase/phosphodiesterase-1; DMEM, Dulbecco's modified Eagle's medium; rP2Y₁₄-R, rat homolog of the P2Y₁₄-R.

Address correspondence to: Dr. T. Kendall Harden, Department of Pharmacology, University of North Carolina, CB#7365 Mary Ellen Jones Bldg., Chapel Hill, NC 27599-7365. E-mail: tkh{at}med.unc.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Arunlakshana O and Schild HO (1959) Some quantitative uses of drug antagonists. Br J Pharmacol Chemother 14: 48–58.

Ault AD and Broach JR (2006) Creation of GPCR-based chemical sensors by directed evolution in yeast. Protein Eng Des Sel 19: 1–8.[Abstract/Free Full Text]

Besada P, Shin DH, Costanzi S, Ko H, Mathe C, Gagneron J, Gosselin G, Maddileti S, Harden TK, and Jacobson KA (2006) Structure-activity relationships of uridine 5'-diphosphate analogues at the human P2Y₆ receptor. J Med Chem 49: 5532–5543.[CrossRef][Medline]

Bodor ET, Waldo GL, Hooks SB, Corbitt J, Boyer JL, and Harden TK (2003) Purification and functional reconstitution of the human P2Y₁₂ receptor. Mol Pharmacol 64: 1210–1216.[Abstract/Free Full Text]

Bogdanov YD, Wildman SS, Clements MP, King BF, and Burnstock G (1998) Molecular cloning and characterization of rat P2Y₄ nucleotide receptor. Br J Pharmacol 124: 428–430.[CrossRef][Medline]

Boyer JL, Siddiqi S, Fischer B, Romero-Avila T, Jacobson KA, and Harden TK (1996) Identification of potent P2Y-purinocepter agonists that are derivatives of adenosine 5'-monophosphate. Br J Pharmacol 118: 1959–1964.[Medline]

Burnstock G (2006) Purinergic signaling. Br J Pharmacol 147: S172–S187.[CrossRef][Medline]

Chambers JK, Macdonald LE, Sarau HM, Ames RS, Freeman K, Foley JJ, Zhu Y, McLaughlin MM, Murdock P, McMillan L, et al. (2000) A G protein-coupled receptor for UDP-glucose. J Biol Chem 275: 10767–10771.[Abstract/Free Full Text]

Communi D, Parmentier M, and Boynaems JM (1996) Cloning, functional expression and tissue distribution of the human P2Y₆ receptor. Biochem Biophys Res Commun 222: 303–308.[CrossRef][Medline]

Communi D, Govaerts C, Parmentier M, and Boynaems JM (1997) Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase. J Biol Chem 272: 31969–31973.[Abstract/Free Full Text]

Coward P, Chan SD, Wada HG, Humphries GM, and Conklin BR (1999) Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors. Anal Biochem 270: 242–248.[CrossRef][Medline]

Cusack NJ and Hourani SM (1982) Competitive inhibition by adenosine 5'-triphosphate of the actions on human platelets of 2-chloroadenosine 5'-diphosphate, 2-azidoadenosine 5'-diphosphate and 2-methylthioadenosine 5'-diphosphate. Br J Pharmacol 77: 329–333.[Medline]

Freeman K, Tsui P, Moore D, Emson PC, Vawter L, Naheed S, Lane P, Bawagan H, Herrity N, Murphy K, et al. (2001) Cloning, pharmacology, and tissue distribution of G-protein-coupled receptor GPR105 (KIAA0001) rodent orthologs. Genomics 78: 124–128.[CrossRef][Medline]

Fumagalli M, Brambilla R, D'Ambrosi N, Volonte C, Matteoli M, Verderio C, and Abbracchio MP (2003) Nucleotide-mediated calcium signaling in rat cortical astrocytes: role of P2X and P2Y receptors. Glia 43: 218–223.[CrossRef][Medline]

Herold CL, Qi AD, Harden TK, and Nicholas RA (2004) Agonist versus antagonist action of ATP at the P2Y₄ receptor is determined by the second extracellular loop. J Biol Chem 279: 11456–11464.[Abstract/Free Full Text]

Houston D, Ohno M, Nicholas RA, Jacobson KA, and Harden TK (2006) [³²P]2-iodo-N⁶-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate ([³²P]MRS2500), a novel radioligand for quantification of native P2Y₁ receptors. Br J Pharmacol 147: 459–467.[CrossRef][Medline]

Houston D, Costanzi S, Jacobson KA, and Harden TK (2008) Development of selective high affinity antagonists, agonists, and radioligands for the P2Y₁ receptor. Combinatorial Chemistry & High-Throughput Screening, in press.

Idzko M, Panther E, Sorichter S, Herouy Y, Berod L, Geissler M, Mockenhaupt M, Elsner P, Girolomoni G, and Norgauer J (2004) Characterization of the biological activities of uridine diphosphates in human dendritic cells: influence on chemotaxis and CXCL8 release. J Cell Physiol 201: 286–293.[CrossRef][Medline]

Ivanov AA, Ko H, Cosyn L, Maddileti S, Besada P, Fricks I, Costanzi S, Harden TK, Van Calenbergh S, and Jacobson KA (2007) Molecular modeling of the human P2Y₂ receptor and design of a selective agonist, 2'-amino-2'-deoxy-2-thiouridine 5'-triphosphate. J Med Chem 50: 1166–1176.[CrossRef][Medline]

Jacobson KA, Costanzi S, Ivanov AA, Tchilibon S, Besada P, Gao ZG, Maddileti S, and Harden TK (2006) Structure activity and molecular modeling analyses of ribose- and base-modified uridine 5'-triphosphate analogues at the human P2Y₂ and P2Y₄ receptors. Biochem Pharmacol 71: 540–549.[CrossRef][Medline]

Kennedy C, Qi AD, Herold CL, Harden TK, and Nicholas RA (2000) ATP, an agonist at the rat P2Y₄ receptor, is an antagonist at the human P2Y₄ receptor. Mol Pharmacol 57: 926–931.[Abstract/Free Full Text]

Ko H, Fricks I, Ivanov AA, Harden TK, and Jacobson KA (2007) Structure activity of uridine 5'-diphosphoglucose (UDP-glucose) analogues as agonists of the human P2Y₁₄ receptor. J Med Chem 50: 2030–2039.[CrossRef][Medline]

Lazarowski ER and Harden TK (1994) Identification of a uridine nucleotide-selective G-protein-linked receptor that activates phospholipase C. J Biol Chem 269: 11830–11836.[Abstract/Free Full Text]

Lazarowski ER, Shea DA, Boucher RC, and Harden TK (2003) Release of cellular UDP-glucose as a potential extracellular signaling molecule. Mol Pharmacol 63: 1190–1197.[Abstract/Free Full Text]

Moore DJ, Chambers JK, Wahlin JP, Tan KB, Moore GB, Jenkins O, Emson PC, and Murdock PR (2001) Expression pattern of human P2Y receptor subtypes: a quantitative reverse transcription-polymerase chain reaction study. Biochim Biophys Acta 1521: 107–119.[Medline]

Müller T, Bayer H, Myrtek D, Ferrari D, Sorichter S, Ziegenhagen MW, Zissel G, Virchow JC Jr, Luttmann W, Norgauer J, et al. (2005) The P2Y₁₄ receptor of airway epithelial cells: coupling to intracellular Ca²⁺ and IL-8 secretion. Am J Respir Cell Mol Biol 33: 601–609.[Abstract/Free Full Text]

Nakahata N and Harden TK (1987) Regulation of inositol trisphosphate accumulation by muscarinic cholinergic and H1-histamine receptors on human astrocytoma cells. Differential induction of desensitization by agonists. Biochem J 241: 337–344.[Medline]

Nicholas RA, Watt WC, Lazarowski ER, Li Q, and Harden TK (1996) Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor. Mol Pharmacol 50: 224–229.[Abstract]

Pendergast W, Yerxa BR, Douglass JG 3rd, Shaver SR, Dougherty RW, Redick CC, Sims IF, and Rideout JL (2001) Synthesis and P2Y receptor activity of a series of uridine dinucleoside 5'-polyphosphates. Bioorg Med Chem Lett 11: 157–160.[CrossRef][Medline]

Qi AD, Kennedy C, Harden TK, and Nicholas RA (2001) Differential coupling of the human P2Y₁₁ receptor to phospholipase C and adenylyl cyclase. Br J Pharmacol 132: 318–326.[CrossRef][Medline]

Roberts VH, Greenwood SL, Elliott AC, Sibley CP, and Waters LH (2006) Purinergic receptors in human placenta: evidence for functionally active P2X₄, P2X₇, P2Y₂, and P2Y₆. Am J Physiol Regul Integr Comp Physiol 290: R1374–R1386.[Abstract/Free Full Text]

Scrivens M and Dickenson JM (2005) Functional expression of the P2Y₁₄ receptor in murine T-lymphocytes. Br J Pharmacol 146: 435–444.[CrossRef][Medline]

Scrivens M and Dickenson JM (2006) Functional expression of the P2Y₁₄ receptor in human neutrophils. Eur J Pharmacol 543: 166–173.[CrossRef][Medline]

Skelton L, Cooper M, Murphy M, and Platt A (2003) Human immature monocyte-derived dendritic cells express the G protein-coupled receptor GPR105 (KIAA0001, P2Y₁₄) and increase intracellular calcium in response to its agonist, uridine diphosphoglucose. J Immunol 171: 1941–1949.[Abstract/Free Full Text]

Warny M, Aboudola S, Robson SC, Sevigny J, Communi D, Soltoff SP, and Kelly CP (2001) P2Y₆ nucleotide receptor mediates monocytes interleukin-8 production in response to UDP or lipopolysaccharide. J Biol Chem 276: 26051–26056.[Abstract/Free Full Text]

Wihlborg AK, Balogh J, Wang L, Borna C, Dou Y, Joshi BV, Lazarowski E, Jacobson KA, Arner A, and Erlinge D (2006) Positive inotropic effects by uridine triphosphate (UTP) and uridine diphosphates (UDP) via P2Y₂ and P2Y₆ receptors on cardiomyocytes and release of UTP in man during myocardial infarction. Circ Res 98: 970–976.[Abstract/Free Full Text]

This article has been cited by other articles:

				I. P. Fricks, R. L. Carter, E. R. Lazarowski, and T. K. Harden Gi-Dependent Cell Signaling Responses of the Human P2Y14 Receptor in Model Cell Systems J. Pharmacol. Exp. Ther., July 1, 2009; 330(1): 162 - 168. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.108.136309v1 325/2/588    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fricks, I. P. Articles by Harden, T. K. Search for Related Content PubMed PubMed Citation Articles by Fricks, I. P. Articles by Harden, T. K. Pubmed/NCBI databases Compound via MeSH Substance via MeSH