Cloning and Functional Expression of a Human Uridine Nucleotide Receptor*

In order to isolate new subtypes of P 2 purinoceptors, sets of degenerate oligonucleotide primers were synthesized on the basis of the best conserved segments in the published sequences of the chick brain P 2Y /P2Y 1 and murine neuroblastoma P 2U /P2Y 2 receptors. Their use in polymerase chain reaction (PCR) experiments on human genomic DNA amplified, among other things, a 712-base pair sequence, that was used as a probe to screen a human genomic DNA library. Several clones corresponding to a single locus were isolated, and the sequence analysis revealed an intronless 1095-base pair open reading frame. The deduced amino acid sequence is consistent with a G protein-coupled receptor and exhibits 51% identity with the human P2Y 2 receptor and 35% with the chick P2Y 1 receptor. A close comparison with the human P2Y 2 sequence reveals the conservation of histidine 262, arginine 265, lysine 289, and arginine 292, which were reported to be involved in nucleotide binding (Erb, L., Garrad, R., Wang, Y., Quinn, T., Turner, J. T., and Weisman, G. A. (1995) J. Biol. Chem. 270, 4185– 4188). Northern blot analysis detected a 1.8-kilobase mes-senger RNA in human placenta. The coding sequence was inserted in the pcDNA3 vector in order to transfect 1321N1 human astrocytoma cells. In cells stably express-ing the receptor, UTP and phages (15) was made for several purified clones which hybridized strongly with the probe. A restriction map and a Southern blotting analysis allowed to isolate a 1.4-kb Nhe I- Eco RV fragment that was subcloned into the pBluescript SK (cid:49) vector (Stratagene). The complete sequence of the new receptor coding sequence was obtained on both strands after subcloning of overlapping fragments in M13mp18 and M13mp19. Cell Culture and Transfection— The P2Y 4 receptor coding sequence was subcloned between the Hin dIII and the Eco RV sites of the pcDNA3 expression vector for transfection into 1321N1 human astrocytoma

In order to isolate new subtypes of P 2 purinoceptors, sets of degenerate oligonucleotide primers were synthesized on the basis of the best conserved segments in the published sequences of the chick brain P 2Y /P2Y 1 and murine neuroblastoma P 2U /P2Y 2 receptors. Their use in polymerase chain reaction (PCR) experiments on human genomic DNA amplified, among other things, a 712base pair sequence, that was used as a probe to screen a human genomic DNA library. Several clones corresponding to a single locus were isolated, and the sequence analysis revealed an intronless 1095-base pair open reading frame. The deduced amino acid sequence is consistent with a G protein-coupled receptor and exhibits 51% identity with the human P2Y 2 receptor and 35% with the chick P2Y 1 receptor. A close comparison with the human P2Y 2 sequence reveals the conservation of histidine 262, arginine 265, lysine 289, and arginine 292, which were reported to be involved in nucleotide binding (Erb, L., Garrad, R., Wang, Y., Quinn, T., Turner, J. T., and Weisman, G. A. (1995) J. Biol. Chem. 270, 4185-4188). Northern blot analysis detected a 1.8-kilobase messenger RNA in human placenta. The coding sequence was inserted in the pcDNA3 vector in order to transfect 1321N1 human astrocytoma cells. In cells stably expressing the receptor, UTP and UDP stimulated the formation of inositol phosphates with equivalent potency and maximal effect, ATP behaved as a partial agonist, and ADP was almost inactive. We have thus cloned a new member of the G protein-coupled P 2 purinergic receptor family, which functionally behaves as a pyrimidinergic receptor.
The cloning of several receptors for ATP has been reported since 1993. In keeping with the latest nomenclature proposal, these P 2 purinergic receptors can be subdivided into two classes: G protein-coupled receptors, or P2Y receptors, and receptors with intrinsic ion channel activity or P2X receptors (2). Two distinct rat P2X receptors have been cloned, respectively, from the vas deferens (3) and pheochromocytoma PC12 cells (4): they have a characteristic topology, with two hydrophobic putatively membrane-spanning segments and an ion pore motif reminiscent of potassium channels. In the P2Y family, the sequences of two subtypes, both coupled to phospholipase C, have been published: chick (5), turkey (6), bovine (7), and mouse and rat (8) P2Y 1 receptors (formerly called P 2Y ); murine (9, 10), rat (11), and human (12) P2Y 2 receptors (previously named P 2U ) on the other hand. In addition, a P2Y 3 receptor, with a preference for ADP over ATP, has been cloned from chick brain, but its sequence is not yet published (13). Furthermore, the 6H1 orphan receptor, cloned from activated chicken T lymphocytes, exhibits a significant degree of homology to the P2Y 1 and P2Y 2 receptors, suggesting that it also belongs to the P2Y family, although its responsiveness to nucleotides has not yet been demonstrated (14). In this paper, we describe the cloning of a new member of the P2Y family, tentatively called P2Y 4 , which exhibits a preference for uridine over adenine nucleotides and therefore constitutes the first example of a pyrimidinergic receptor.
Cloning and Sequencing-Degenerate oligonucleotide primers were synthesized on the basis of the best conserved segments between the murine P2Y 2 and the chick P2Y 1 receptor sequences. These primers were used to amplify novel receptor gene fragments by low stringency PCR starting from human genomic DNA. The amplification conditions were as follows: 93°C, 1 min, 50°C, 2 min, 72°C, 3 min; 35 cycles. The PCR products with sizes compatible with P 2 receptor gene fragments were subcloned in M13mp18 and M13mp19 and sequenced by the Sanger dideoxy nucleotide chain termination method. One of the resulting clones sharing similarities with P 2 receptors was labeled by random priming and used to screen a human genomic DNA library constructed in the Charon 4a vector. The hybridization was in 6 ϫ SSC (1 ϫ SSC: 0.15 M NaCl, 0.015 M sodium citrate) and 40% formamide at 42°C for 14 h, and the final wash conditions were 0.1 ϫ SSC, 0.1% SDS at 65°C. A preparation of phages (15) was made for several purified clones which hybridized strongly with the probe. A restriction map and a Southern blotting analysis allowed to isolate a 1.4-kb NheI-EcoRV fragment that was subcloned into the pBluescript SK ϩ vector (Stratagene). The complete sequence of the new receptor coding sequence was obtained on both strands after subcloning of overlapping fragments in M13mp18 and M13mp19.
Cell Culture and Transfection-The P2Y 4 receptor coding sequence was subcloned between the HindIII and the EcoRV sites of the pcDNA3 expression vector for transfection into 1321N1 human astrocytoma * This work was supported by an Action de Recherche Concertée of the Communauté Française de Belgique, by the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Federal Service for Science, Technology and Culture, by a grant of the Fonds de la Recherche Scientifique Médicale, by the Fonds Médical Reine Elisabeth, and by Boehringer Ingelheim. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) X91852.
§ Chargé de Recherche of the Fonds National de la Recherche Scientifique.
¶ Maître de Recherche of the Fonds National de la Recherche Scientifique. cells, a cell line which does not respond to nucleotides and which has already been used for the expression of purinergic receptors (6,12). Cells were transfected with the recombinant pcDNA3 plasmid using the calcium phosphate precipitation method as described (16). 1321N1 cells were incubated for 6 h at 37°C in the presence of pcDNA3 vector alone or vector containing the P2Y 4 receptor coding sequence, then washed, and incubated in culture medium (10% FCS, 100 units/ml penicillin, 100 g/ml streptomycin, and 2.5 g/ml amphotericin B in Dulbecco's modified Eagle's medium). The selection with G418 (400 g/ml) was started 2 days after transfection. From the pool of transfected 1321N1 cells, individual clones were isolated by limiting dilution with the aim of selecting clones with high IP stimulation factors in response to nucleotides. The different clones were maintained in a medium containing 400 g/ml G418.
Inositol Phosphates (IP) Measurement-1321N1 cells were labeled for 24 h with 10 Ci/ml [ 3 H]inositol in inositol-free Dulbecco's modified Eagle's medium containing 5% FCS, antibiotics, amphotericin, and 2 units/ml apyrase as described (6). Cells were washed twice with Krebs-Ringer Hepes (KRH) buffer of the following composition (124 mM NaCl, 5 mM KCl, 1.25 mM MgSO 4 , 1.45 mM CaCl 2 , 1.25 mM KH 2 PO 4 , 25 mM Hepes (pH 7.4), and 8 mM glucose) and incubated in the same medium supplemented with 10 mM LiCl for 5 min before the addition of the agonists. The incubation was stopped after 20 min by the addition of an ice-cold 3% perchloric acid solution. IP were extracted and separated on Dowex columns as described previously (17).
Northern Blot and Southern Blot Analysis-Total and poly(A) ϩ RNA were prepared from different tissues and human cell lines using the guanidinium thiocyanate-cesium chloride procedure (15), denatured by glyoxal and fractionated by electrophoresis on a 1% agarose gel in 10 mM phosphate buffer, pH 7.0. DNA samples, prepared from the Charon 4a clones, were digested with restriction enzymes. Northern and Southern blots were prepared (15) and baked for 90 min at 80°C. Membranes were prehybridized for at least 4 h and hybridized overnight with the same probe as for the screening, at 42°C in a solution containing 50% formamide for Northern blots and 40% formamide for Southern blots. Filters were washed twice for 15 min in 2 ϫ SSC at room temperature and then twice for 30 min in 0.2 ϫ SSC at 60°C before being exposed at Ϫ70°C in the presence of intensifying screens for 5 days (Northern blots) or 1 h (Southern blots).

RESULTS
Cloning and Sequencing-In order to isolate new subtypes of P 2 receptors, sets of degenerate oligonucleotides primers were synthesized on the basis of the best conserved segments in the published sequences of the chick brain P2Y 1 (5) and murine neuroblastoma P2Y 2 (9) receptors. These primers were used in low-stringency PCR on human genomic DNA as described (18). Some combinations generated discrete bands with a size compatible with that expected for P 2 receptors. The partial sequences obtained after sequencing were translated into peptidic sequences and compared to a local databank which contains G protein-coupled receptor sequences. Most of the clones resulting from these PCR products encoded a part of a new receptor which displayed 58% identity with the murine P2Y 2 receptor and 42% identity with the chick P2Y 1 receptor partial sequences. In addition, some clones encoded a peptidic sequence presenting 87% identity with the chick P2Y 1 receptor and are therefore believed to represent fragments of the human P2Y 1 gene (data not shown).
The partial sequence of the new receptor was used as a probe to screen a human genomic DNA library. Several clones that strongly hybridized with the probe at high stringency conditions were obtained and purified. The inserts of the clones varied from 12 to 17 kb, and restriction analysis revealed that all clones belonged to a single locus. The full sequence of a 1.4-kb NheI-EcoRV fragment was obtained, and an intronless open reading frame of 1095 bp was identified (Fig. 1). The predicted molecular mass of the encoded protein is 36.5 kDa. This molecular mass is unlikely to be modified in vivo, since no N-glycosylation consensus sequences are found in the putative exofacial regions. In contrast with the human P2Y 2 receptor, there is no RGD motif, an integrin binding consensus sequence, in the putative first extracellular loop. Some potential sites of phosphorylation by protein kinase C or by calmodulin-dependent protein kinases were identified in the third intracellular loop and in the carboxyl-terminal part of the receptor (Fig. 1). The four positively charged amino acids which have been reported to play a role in the P2Y 2 receptor activation by ATP and UTP (1) are conserved in the P2Y 4 sequence: His 262 , Arg 265 , Lys 289 , and Arg 292 (Fig. 1). The P2Y 4 amino acid sequence was compared to the chick P2Y 1 and the murine P2Y 2 amino acid sequences and to their closest neighbors in the G proteincoupled receptor family (Fig. 2). It is clear that, from a structural point of view, the newly cloned receptor is more closely related to the human P2Y 2 receptor (51% of identity between the complete sequences) than to the chick P2Y 1 receptor (35%).
Tissue Distribution of the P2Y 4 Receptor-The tissue distribution of P2Y 4 transcripts was investigated by Northern blotting. A number of rat tissues (heart, brain, liver, testis, and kidney) were tested using a human probe at low stringency, but no hybridization signal could be obtained. No P2Y 4 transcript could be detected in the following human cell lines: K562 leukemia cells (Fig. 3) and HL-60 leukemia cells and SH-SY5Y human neuroblastoma cells (data not shown). On the contrary, a strong signal, corresponding to a 1.8-kb mRNA, was found in human placenta (Fig. 3).
Functional Expression of the New P 2 Receptor in 1321N1 Cells-After transfection of the pcDNA3-P2Y 4 construction in 1321N1 cells, the pool of G418-resistant clones was tested for Human Uridine Nucleotide Receptor 30850 their functional response (IP 3 accumulation) to ATP and UTP. Both nucleotides were found to be agonists of the P2Y 4 receptor, but the response to UTP was more robust; as previously reported (6,12), none of the tested nucleotides (UTP, UDP, ATP, 2-methylthio-ATP, and ADP) produced a detectable effect in cells transfected with the pcDNA3 vector, whereas carbachol elicited strong responses (data not shown). About 20 transfected clones were then isolated and tested for their response to UTP. The clone presenting the highest IP stimulation factor in response to UTP was selected and used in all subsequent experiments (13 experiments in all). UTP and UDP produced the same maximal effect, with a similar potency (EC 50 around 2 M) (Fig. 4). ATP had a lower potency (EC 50 around 20 M), and its maximal effect was only 35% of that of UTP and UDP (mean of four independent experiments). The effect of ADP was barely detectable (Fig. 4), and 2-methylthio-ATP was completely inactive (data not shown). DISCUSSION We describe here the cloning of a human gene encoding a novel member of the P 2 purinergic receptor family. This receptor was provisionally named P2Y 4 : this assignment is tentative since we are aware of other recently cloned subtypes might qualify for number 4. No N-glycosylation consensus sequence can be detected in the putative exofacial regions. This situation is rare in G protein-coupled receptors but not unique: it has also been reported for the rat ␣ 2B -adrenergic receptor (19), a human receptor for the LD78 chemokine (20) and the rat RBS11 orphan receptor (21). Despite its structural relatedness to the human P2Y 2 receptor, the newly cloned receptor exhibits one conspicuous difference in the putative first extracellular loop: the RGD motif, an integrin binding consensus sequence, present in the P2Y 2 receptor is not conserved, although clusters of four amino acids are perfectly conserved on both sides. Sitedirected mutagenesis studies have demonstrated the crucial influence of charged amino acids in transmembrane helices 6 and 7 of the P2Y 2 receptor on agonist potency and specificity (1). His 262 , Arg 265 , and Arg 292 seem to be directly involved in the binding of the negatively charged phosphate groups, whereas a mutation of Lys 289 into Arg decreases the affinity for ATP/UTP and increases that for ADP/UDP. These four residues are conserved in the P2Y 4 subtype, whereas Arg 265 and Lys 289 are replaced respectively by Lys and Gln in the P2Y 1 receptor (5). The P2Y 4 receptor sequence presents also a series of 10 amino acids (LFLTCISVHR) which is a consensus sequence between all the P2Y receptors cloned until now. Potential sites of phosphorylation by protein kinase C or by calmodulin-dependent protein kinases were identified in the third intracellular loop and in the carboxyl-terminal part of the receptor. These regions are also particularly rich in serine and threonine residues that could be targets for the family of G protein-coupled receptor kinases. A strong signal corresponding to a 1.8-kb mRNA was detected in human placenta. No other human organ has been tested so far. Various rat tissues tested at low stringency and several human cell lines were negative. This suggests a rather restricted expression of the P2Y 4 receptor.
In the late eighties, it became apparent that extracellular uridine nucleotides exert effects on many tissues and cells. It was then proposed that these actions are mediated by pyrimidinoceptors distinct from the purinoceptors involved in the response to adenine nucleotides (22). The existence of nucleotide receptors common to ATP and UTP constituted an alternative possibility, in favor of which experimental evidence started to accumulate, for instance the lack of additivity and cross-desensitization of the responses to the two nucleotides (23,24). The final demonstration of this concept was provided by the cloning and functional expression of the P 2U /P2Y 2 receptor at which ATP and UTP are equally active and equipotent (9,10,12). However the pyrimidinoceptor hypothesis reemerged with the observation that UTP and UDP stimulate inositol phosphates formation in C6 -2B rat glioma cells, whereas ATP and ADP are totally inactive (25). However, the C6 glioma cells, from which the C6 -2B subclone was isolated, express a typical P 2U response: a mutation of that receptor, with selective loss of the adenine nucleotides responsiveness, Human Uridine Nucleotide Receptor 30851 might thus explain the peculiar behavior of C6 -2B cells. Our results conclusively demonstrate the existence of a human gene encoding a receptor with a preference for pyrimidine over purine nucleotides. Indeed, at the P2Y 4 receptor, UTP and UDP were full agonists, whereas ATP behaved as a partial agonist with a lower affinity than UTP and ADP was barely active. However, on the basis of its structure, this receptor clearly belongs to the P2Y family, which thus encompasses selective purinoceptors (P2Y 1 ), nucleotide receptors responsive to both adenine and uridine nucleotides (P2Y 2 ), and pyrimidinoceptors (P2Y 4 ).