Introduction

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P2Y receptors are G protein-coupled receptors that mediate a wide variety of physiological effects in response to extracellular adenine and uridine nucleotides (Harden et al., 1995; Filtz et al., 1997). Although the existence of multiple subtypes of P2Y receptors was evident from early pharmacological studies, the application of molecular cloning and expression of P2Y receptors has provided a better understanding of this family of nucleotide receptors. Eleven different G protein-coupled receptors (termed P2Y₁ through P2Y₁₁) have been claimed to be members of the P2Y receptor family, but only five of these receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁) have been cloned from mammalian species and shown unambiguously to mediate nucleotide-promoted second messenger responses. The P2Y₁ receptor is activated specifically by adenine nucleotides, with ADP being more potent than ATP (Henderson et al., 1995; Schachter et al., 1996), although recent studies have suggested that ATP and ATP derivatives are antagonists at the P2Y₁ receptor (Leon et al., 1997; Hechler et al., 1998). The P2Y₂ receptor is activated equipotently by ATP and UTP (Parr et al., 1994), the P2Y₄ receptor is activated only by UTP (Nguyen et al., 1995; Communi et al., 1996; Lazarowski et al., 1997), the P2Y₆ receptor is activated selectively by UDP (Nicholas et al., 1996), and the P2Y₁₁ receptor is activated primarily by ATP (Communi et al., 1997). All of these receptors activate PLC. In addition to coupling to PLC, the P2Y₁₁ receptor has been reported to activate adenylyl cyclase (Communi et al., 1997).

The cloning from a chick brain cDNA library and characterization of an additional P2Y receptor, termed the p2y3 receptor,¹ was reported recently (Webb et al., 1996). The p2y3 receptor is 65% identical to the rat P2Y₆ receptor and 38, 42, and 41% identical to human P2Y₁, P2Y₂, and P2Y₄ receptors, respectively. The nucleotide selectivity and second messenger signaling properties of the p2y3 receptor have not been completely defined. In studies measuring the amplitudes of Ca²⁺-activated Cl currents in p2y3 receptor-expressing Xenopus laevis oocytes, ADP (100 µM) elicited the largest current, followed by ATP--thiol, UTP, ATP, and UDP. In contrast, UDP was the most potent agonist, followed by UTP, ADP, ATP--thiol, and ATP, in measurements of intracellular Ca²⁺ mobilization in p2y3 receptor-expressing Jurkat cells. Although it is likely that the mobilization of intracellular Ca²⁺ was a consequence of activation of PLC and production of inositol-1,4,5-trisphosphate, no direct analysis of inositol lipid hydrolysis was reported.

Webb et al. (1996) proposed that the chick p2y3 receptor is a unique subtype of P2Y receptor. However, given the similarities in both sequence identity and apparent nucleoside diphosphate selectivity of the chick p2y3 receptor and the rat P2Y₆ receptor, it is conceivable that the p2y3 receptor is the avian homologue of the previously reported mammalian P2Y₆ receptor. To distinguish between these two possibilities and to directly compare the signaling properties of these two receptors, we have expressed both the turkey p2y3 receptor and the rat P2Y₆ receptor in 1321N1 cells and have determined their agonist selectivities and second messenger signaling properties under identical conditions. In addition, we have screened at low stringency two human genomic libraries and a Southern blot with a turkey p2y3 probe, to identify a possible human homologue. Our results indicate that mammalian P2Y₆ and avian p2y3 receptors both activate PLC and that the receptors have similar but not identical agonist selectivities. Moreover, no receptor apparently exists in the human genome with greater homology to the avian p2y3 receptor than the human P2Y₆ receptor. We conclude from these data that the chick p2y3 receptor is the avian homologue of the mammalian P2Y₆ receptor.

	Experimental Procedures

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Materials. ATP, ADP, UTP, and UDP were purchased from Pharmacia (Piscataway, NJ). The purity of these nucleotides was estimated to be >99% by HPLC analysis. 2-Methylthio-ATP, 2-methylthio-ADP, and ADP--thiol were purchased from Research Biochemicals (Natick, MA). 5-Bromo-UTP was from Sigma Chemical Co. (St. Louis, MO), and 5-bromo-UDP was synthesized by incubating 5-bromo-UTP with hexokinase and glucose (Lazarowski et al., 1996). myo-[³H]Inositol was from American Radiolabeled Chemicals (St. Louis, MO). [-³²P]dATP was from New England Nuclear. Hexokinase was from Sigma. All tissue culture reagents were from the tissue culture facility at the Lineberger Comprehensive Cancer Center (University of North Carolina).

Cloning and expression of the turkey p2y3 receptor. Because of the similarity between chick and turkey genes, the turkey p2y3 receptor was amplified from turkey genomic DNA by PCR with primers based on the published chick p2y3 receptor sequence (Filtz et al., 1994; Webb et al., 1996). The upstream and downstream oligonucleotides were 5'-GAGACTCGAGCCACCATGAGCATGGCCAACTTCACGGG-3' (complementary to bases 1-23 of the chick p2y3 receptor coding sequence, with the ATG codon in bold type) and 5'-GAGAGGATCCCATCCCCATCTCCGCACCATG-3' (complementary to bases 60-81 downstream of the stop codon of the chick p2y3 receptor coding sequence), respectively. The entire coding sequence of the turkey p2y3 receptor was amplified from 1 µg of turkey genomic DNA with Pyrococcus furiosus DNA polymerase (Stratagene, La Jolla, CA), using the following amplification conditions: 94° for 30 sec, 58° for 30 sec, and 72° for 1 min for 35 cycles. The amplified p2y3 receptor gene was subcloned into the HpaI site of pLXSN, and clones harboring the insert in the correct orientation were identified by restriction mapping. Recombinant viral particles were produced in PA317 cells and used to infect 1321N1 cells as described previously (Comstock et al., 1997). 1321N1 cells expressing the rat P2Y₆ receptor were produced in a similar manner (Nicholas et al., 1996), except that a single clone was expanded from the cell population and used in activity assays. Cells were selected with 1 mg/ml G418 in Dulbecco's modified Eagle medium and were maintained in the same medium with 400 µg/ml G418.

Measurement of [³H]inositol phosphate accumulation. Cells were plated in 24-well plates at 5 × 10⁴ cells/well and were assayed after 3 days in culture. Inositol lipids were radiolabeled by overnight incubation with 0.4 µCi of myo-[³H]inositol/well, in 200 µl of inositol-free Dulbecco's modified Eagle medium. No changes of medium were made after the addition of [³H]inositol. Drug challenges were initiated by addition of 50 µl of 5-fold concentrated agonists in 50 mM LiCl. After a 5-min incubation at 37°, the reactions were stopped by aspiration of the medium and addition of 0.5 ml of boiling 10 mM EDTA, pH 8.0. [³H]Inositol phosphates were isolated by chromatography on Dowex AG1-X8 resin, as described previously (Filtz et al., 1994; Lazarowski et al., 1995). All nucleoside diphosphates were treated with hexokinase, as described (Nicholas et al., 1996), to remove contaminating nucleoside triphosphates.

Measurement of intracellular Ca²⁺ levels. 1321N1 cells expressing either the turkey p2y3 receptor or the rat P2Y₆ receptor were plated at low density on glass coverslips and assayed after 2-3 days in culture. On the day of the assay, the cells were incubated for 30 min with 0.5 µM fura-2/acetoxymethyl ester, washed, placed in a sealed recording chamber, and continuously superfused with Hanks' buffered saline solution at a constant flow rate of 1.4 ml/min (Palmer et al., 1994). To initiate responses, cells were superfused for 30 sec with UTP and UDP (freshly purified by HPLC). Changes in intracellular Ca²⁺ levels were quantified by recording the fluorescence of 8-12 cells at 510 nm, after excitation at 340 and 380 nm. No more than 25 cells were in the visual field in any single experiment. The fluorescence ratio values from individual cells were averaged for each concentration and plotted versus time, and the area under the curve was determined by using Prism (GraphPad) software.

Screening of human genomic libraries with a turkey p2y3 probe. Two genomic libraries were screened with a turkey p2y3 receptor probe, in an attempt to identify a human homologue of the p2y3 receptor. One of the libraries was purchased from Stratagene (a -fix library of placental DNA with insert sizes of 9-23 kilobases), and the other was constructed by Dr. John Lowe (University of Michigan, Ann Arbor, MI) (a -fix library of DNA isolated from peripheral blood lymphocytes, with insert sizes of 9-15 kilobases). For each screening, 1 × 10⁶ phage were plated and transferred in duplicate to nylon filters. The filters were hybridized at 42° in 4.5× SSC (1× = 150 mM NaCl, 15 mM sodium citrate), 50% formamide, 1× Denhardt's solution (1× = 0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin), and 0.1% SDS, with a ³²P-labeled probe encompassing the entire coding sequence of the turkey p2y3 receptor. The filters were washed three times, for 15 min each, in 0.5× SSC/0.1% SDS at 50° and were apposed to film for 2-4 days. All plaques displaying hybridization on both sets of filters were isolated and plaque-purified. The identity of each positively hybridizing plaque was determined by a combination of high-stringency screening (wash conditions of 0.1× SSC/0.1% SDS at 70°) and PCR with P2Y receptor-specific primers.

	Results

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Direct comparison of the agonist selectivities of rat P2Y₆ and turkey p2y3 receptors expressed in 1321N1 cells. The turkey homologue of the chick p2y3 receptor was amplified from genomic DNA using primers based on the chick sequence. The amino acid sequence of the turkey p2y3 receptor, like many other turkey signaling proteins, was nearly identical (97.6%) to that of its chick homologue, with eight amino acid differences between the two (GenBank accession number AF069555). Because the pharmacological selectivities and second messenger signaling properties of the chick p2y3 receptor and the rat P2Y₆ receptor have not been directly compared, the turkey p2y3 and rat P2Y₆ receptors were expressed in 1321N1 cells and assayed under identical conditions, as described in Experimental Procedures. As predicted from the results of Webb et al. (1996), who measured mobilization of intracellular Ca²⁺ in Jurkat cells, both UTP and UDP stimulated inositol phosphate accumulation in 1321N1 cells stably expressing the turkey p2y3 receptor (see below). Therefore, the avian p2y3 receptor, like the mammalian P2Y₆ receptor, activates PLC.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Pharmacological selectivities of the turkey p2y3 and rat P2Y6 receptors for uridine nucleotides. Inositol phosphate accumulation was measured in 1321N1 cells expressing either the turkey p2y3 receptor (A) or the rat P2Y6 receptor (B), after a 5-min incubation with the indicated nucleotides in the presence of LiCl. Nucleoside diphosphates were pretreated with hexokinase and glucose as described previously (Nicholas et al., 1996). The data were normalized to the maximal effect of UDP in each experiment and represent results from at least three different experiments. The standard error of each value was always <10% of the mean. The values for basal and agonist-induced [3H]inositol phosphate accumulation were ~600 and ~2700 cpm, respectively, for p2y3 receptor-expressing 1321N1 cells and ~900 and ~3800 cpm, respectively, for P2Y6 receptor-expressing 1321N1 cells. 5BrUDP, 5-bromo-UDP.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Promotion of intracellular Ca2+ mobilization by HPLC-purified UTP and UDP in 1321N1 cells expressing the turkey p2y3 receptor (A) or the rat P2Y6 receptor (B). Cells were plated on glass coverslips at low density and were loaded with fura-2/acetoxymethyl ester on the day of the experiment. The cells were placed in a sealed chamber under constant superfusion, and intracellular Ca2+ levels were measured in response to a 30-sec application of HPLC-purified nucleotides. The 340/380-nm ratios for 10-15 cells in a single optical field for each concentration of agonist were averaged and plotted versus time, to yield the area under the curve.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Pharmacological selectivities of the turkey p2y3 and rat P2Y6 receptors for adenine nucleotides and nucleotide derivatives. Inositol phosphate accumulation was measured in 1321N1 cells expressing either the turkey p2y3 receptor (A) or the rat P2Y6 receptor (B), after a 5-min incubation with the nucleotides in the presence of LiCl. Nucleoside diphosphates were pretreated with hexokinase and glucose as described previously (Nicholas et al., 1996). The data were normalized to the maximal effect of UDP in each experiment and represent results from at least three different experiments. The standard error of each value was always <10% of the mean. 2MeSADP, 2-methylthio-ADP; ADPS, ADP--thiol.

Attempts to clone a human homologue of the avian p2y3 receptor. Unambiguous confirmation that the p2y3 receptor is a unique P2Y receptor subtype would follow from the cloning of its mammalian homologue. Therefore, we devised a strategy to identify a potential human homologue of the turkey p2y3 receptor, based on low-stringency screening of human genomic libraries with a p2y3 receptor probe. Genomic libraries were chosen for this purpose because several genome equivalents could be sampled in a single screening, no prior knowledge of tissue distribution was required, and the issue of clone representation in cDNA libraries could be avoided. We reasoned that, if a mammalian homologue of the avian p2y3 receptor exists, it would be more homologous to a turkey p2y3 probe than to a rat P2Y₆ receptor probe. Therefore, hybridization and washing conditions were optimized for filters containing P2Y₁, P2Y₂, P2Y₄, and P2Y₆ receptor genomic bacteriophage  clones (previously isolated from the leukocyte genomic library), using a turkey p2y3 receptor probe. This optimization of screening parameters identified conditions that ensured clear identification of human P2Y₆ clones during library screening.

View larger version (68K): [in this window] [in a new window]   Fig. 4.   Southern blots of human genomic DNA screened with either a turkey p2y3 receptor (right) or rat P2Y6 receptor (left) probe. Southern blots of human genomic DNA were prepared and hybridized with full-length probes derived from either the turkey p2y3 or rat P2Y6 receptor. The blot hybridized with the rat P2Y6 receptor probe was washed at high stringency and the blot hybridized with the turkey p2y3 receptor probe was washed at low stringency, as described in Experimental Procedures. The extra bands observed in the control lanes of the blot hybridized with the rat P2Y6 receptor probe were likely the result of hybridization of the probe with vector sequences (i.e., the probe contained a small amount of vector DNA).

	Discussion

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Webb et al. (1996) reported the cloning and characterization of a chick P2Y receptor that is 65% identical to the rat P2Y₆ receptor. We have cloned the turkey homologue (~98% identical to the chick sequence) of the chick p2y3 receptor and expressed it in 1321N1 cells, to directly compare its pharmacological selectivity with that of the rat P2Y₆ receptor. These studies show that the two receptors have very similar, but not identical, pharmacological selectivities. Both the turkey p2y3 and rat P2Y₆ receptors were activated most potently by UDP and much less potently by UTP. Similarly, 5-bromo-UDP was equipotent with UDP at both receptors. In contrast to the similar effects of uridine nucleotides, adenine nucleotides were more potent at the turkey p2y3 receptor than at the rat P2Y₆ receptor. The pharmacological selectivity of the turkey p2y3 receptor for stimulation of inositol lipid hydrolysis in 1321N1 cells was very similar to that described for the chick p2y3 receptor for mobilization of intracellular Ca²⁺ in Jurkat cells but very different from the selectivity reported for the chick receptor expressed in X. laevis oocytes. The source of this difference in apparent agonist selectivity in these expression systems is not clear.

The status of UTP as an agonist at the p2y3 and P2Y₆ receptors also was assessed. The stimulatory activity observed during incubation with UTP potentially could have resulted entirely from breakdown of UTP to UDP by ecto-nucleotidases or from contamination of the UTP stocks with UDP. Therefore, we took advantage of a constant flow system for measurement of intracellular Ca²⁺ levels in cells plated at low density, to eliminate the contribution of ecto-nucleotidase activity during determination of nucleotide selectivities. HPLC-purified UTP was a full agonist, with ~50-fold lower potency than UDP, in 1321N1 cells expressing either the turkey p2y3 or rat P2Y₆ receptor.

We reported previously that ADP was a partial agonist at the rat P2Y₆ receptor (Nicholas et al., 1996), whereas in this study it was apparently a full agonist. The agonist effects of ADP reported here may be the result of higher receptor expression levels in the clonal cell line used in this study, compared with the population of cells assayed previously. Higher receptor densities could result in full-agonist effects of partial agonists and in apparent increases in the potencies of full agonists (Harden et al., 1997). This possibility is supported by the nearly 10-fold lower EC₅₀ of UDP for stimulation of inositol lipid hydrolysis in the clonal line of P2Y₆ receptor-expressing cells, compared with the value previously observed with the cell population.

Webb et al. (1996) proposed that the avian p2y3 receptor is a novel P2Y receptor subtype. This conclusion was based on the assumption that the 65% sequence identity of the chick p2y3 receptor to the rat P2Y₆ receptor was too low for these receptors to be species homologues. However, this percent identity is much greater that the percent identity (~40%) of the p2y3 receptor to other mammalian P2Y receptors. Furthermore, the percent identity between the two receptors increases to 74% when only the putative membrane-spanning regions are considered. The avian p2y3 receptor and mammalian P2Y₆ receptor have similar pharmacological selectivities, and we observed no evidence for the existence of a gene in the human genome that is more homologous to the turkey p2y3 receptor than the human P2Y₆ receptor. The most parsimonious interpretation of these results is that the avian p2y3 and human P2Y₆ receptors are species homologues.

There are parallels between the P2Y and -AR subtypes in both birds and mammals. Turkeys have three known -AR subtypes. The turkey ₂-AR is 85% identical to its mammalian orthologue and exhibits nearly identical pharmacological selectivity (Del Toro F and Nicholas RA, manuscript in preparation), the turkey ₁-AR is 68% identical to the mammalian ₁-AR and has similar but not identical pharmacological selectivity (Minneman et al., 1980; Yarden et al., 1986), and the turkey _4C-AR is approximately 50% identical to all known -AR subtypes and has unique pharmacological selectivity (Chen et al., 1994). No mammalian homologue of the _4C-AR has yet been identified. A nearly identical comparison can be made for avian and mammalian P2Y receptors. Thus, the avian and mammalian P2Y₁ receptors are 85% identical and exhibit nearly identical pharmacological selectivities (Schachter et al., 1996), the avian p2y3 and mammalian P2Y₆ receptors are 65% identical and exhibit similar but not identical pharmacological selectivities, and a newly identified avian P2Y receptor (Boyer et al., 1997) has 40-50% identity to the four mammalian P2Y receptor subtypes and pharmacological selectivity similar to that of the P2Y₂ receptor. As with the _4C-AR, no mammalian homologue for this avian P2Y receptor has been identified.