Patent Abstract:
the present invention concerns a new receptor having a preference for pyrimidine nucleotides , preferably utp , or purine nucleotides and udp , and which has an amino acid sequence having more than 60 % homology with the amino acid sequence shown in fig . 1 .

Detailed Description:
trypsin was from flow laboratories ( bioggio , switzerland ) and the culture media , reagents , g418 , fetal calf serum ( fcs ), restriction enzymes and taq polymerase were purchased from gibco brl ( grand island , n . y .). the radioactive products myo - d -[ 2 - 3 h ] inositol ( 17 . 7 ci / mmol ) and [ a 32 p ] atp ( 800 ci / mmol ) were from amersham ( gent , belgium ). dowex ag1x8 ( formate form ) was from bio - rad laboratories ( richmond , calif .). utp , udp , atp , adp , carbachol , licl and apyrase grade vii were obtained from sigma chemical co . ( st . louis , mo .). 2mesatp was from research biochemicals inc . ( natick , mass .). pcdna3 is an expression vector developed by invitrogen ( san diego , calif .). degenerate oligonucleotide primers were synthesized on the basis of the best conserved segments between the murine p2y2 and the chick p2y1 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 p2 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 p2 receptors , was labelled by random priming and used to screen a human genomic dna library constructed in the λ charon 4a vector . the hybridization was in 6 × ssc ( 1 × 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 × ssc , 0 . 1 % sds at 65 ° c . a preparation of λ phages ( 15 ) was made for several 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 − vector ( stratagene ). the complete sequence of a new receptor coding sequence was obtained on both strands after subcloning of overlapping fragments in m13mp18 and m13mp19 . 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 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 ( pcdna3 - p2y 4 ) using the calcium phosphate precipitation method as described ( 16 ). 1321n1 cells were incubated for 6 hours 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 u / ml penicillin , 100 μg / ml streptomycin and 2 . 5 μg / ml amphotericin b in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem )). the selection with g418 ( 400 μg / ml ) was started two 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 μg / ml g418 . 1321n1 cells were labelled for 24 hours with 10 μci / ml [ 3 h ] inositol in inositol - free dmem ( dulbecco &# 39 ; s modified eagle &# 39 ; s medium ) medium containing 5 % fetal calf serum , 100 u / ml penicillin , 100 μg / ml streptomycin , 2 . 5 μg / ml amphotericin b and 400 μg / ml g418 . cells were washed twice with krh ( krebs - ringer hepes ) buffer of the following composition ( 124 mm nacl , 5 mm kcl , 1 . 25 mm mgsc 4 , 1 . 45 mm cacl 2 , 25 mm hepes ( ph 7 . 4 ) and 8 mm glucose ) and incubated in this medium for 30 min . the agonists were added in the presence of licl ( 10 mm ) and the incubation was stopped after 30 s , 5 min or 20 min by the addition of an ice - cold 3 % perchloric acid solution . for the time course study , licl ( 10 mm ) was added 5 min before the agonists and the incubation was stopped at different times . when tested , pertussis toxin ( 20 ng / ml ) was added for 18 h during the labelling period time and during the stimulation by the agonist . inositol phosphates were extracted and insp 3 was isolated by chromatography on dowex column as described previously ( 17 ). binding assays of [ α 32 p ] utp to cell membranes were carried out in tris - hcl ( 50 mm , ph 7 . 5 ), edta 1 mm in a final volume of 0 . 5 ml , containing 25 - 50 μg of protein and 0 . 5 nm of radioligand ( 27 ). the assays were conducted at 30 ° c . for 5 min . incubations were stopped by the addition of 4 ml of ice - cold tris - hcl ( 50 mm , ph 7 . 5 ) and rapid filtration through whatman gf / b filters under reduced pressure . the filters were then washed three times with 2 ml of the same ice - cold tris - hcl buffer . radioactivity was quantified by liquid scintillation counting , after an overnight incubation of the filters in liquid scintillation mixture . total and poly ( a ) + 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 λ 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 hours 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 × ssc at room temperature and then twice for 30 min in 0 . 2 × ssc at 60 ° c . before being exposed at − 70 ° c . in the presence of intensifying screens for 5 days ( northern blots ) or 1 hour ( southern blots ). in order to isolate new subtypes of p2 receptors , sets of degenerate oligonucleotide primers were synthesized on the basis of the best conserved segments in the published sequences of the chick brain p2y1 ( 5 ) and murine neuroblastoma p2y2 ( 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 p2 receptors . for example , the primer [ 5 ′- cagatctagata ( ct ) atgtt ( ct )( ac ) a ( ct )( ct ) t ( acgt ) gc - 3 ] corresponding to the second transmembrane region and the primer [ 5 ′- tcttaagcttgg ( ag ) tc ( acgt ) a ( cg )( ag ) ca ( ag ) ct ( ag ) tt - 3 ′] corresponding to the seventh transmembrane region amplified a 712 bp fragment . 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 p2y2 receptor and 42 % identity with the chick p2y1 receptor partial sequences . in addition , some clones encoded a peptidic sequence presenting 87 % identity with the chick p2y1 receptor and are therefore believed to represent fragments of the human p2y1 gene . 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 . the sequence is depicted in fig1 where the putative membrane - spanning domains are underlined and numbered i to vii . the predicted molecular weight of the encoded protein is 36 . 5 kda . this molecular weight 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 p2y2 receptor , there is no rgd motif , an integrin binding consensus sequence , in the putative first extracellular loop . the three amino acid ( ahn ) corresponding to the rgd sequence in the first extracellular loop of the p2y2 receptor are represented in bold in fig1 . some potential sites of phosphorylation by protein kinase c ( pkc ) or by calmodulin - dependent protein kinases were identified in the third intracellular loop and in the carboxyterminal part of the receptor . the putative phosphorylation sites by pkc or by calmodulin - dependent protein kinases and pkc are indicated respectively by black squares and by open circles in fig1 . the four positively charged amino acid which have been reported to play a role in the p2y2 receptor activation by atp and utp ( 1 ) are conserved in the p2y4 sequence : his 262 , arg 265 , lys 289 and arg 292 ( fig1 ). the p2y4 amino acid sequence was compared to the chick p2y1 and the murine p2y2 amino acid sequences and to their closest neighbours in the g protein - coupled receptor family ( fig2 ). the plot was constructed using the multiple sequence alignment program pileup of the gcg package ( 26 ). for each sequence , the analysis takes into account a segment covering the first five putative membrane - spanning domains . it is clear that , from a structural point of view , the newly cloned receptor is more closely related to the human p2y2 receptor ( 51 % of identity between the complete sequences ) than to the chick p2y1 receptor ( 35 %). the tissue distribution of p2y4 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 p2y4 transcript could be detected in the following human cell lines : k562 leukemia cells ( fig3 ), hl - 60 leukemia cells and sh - sy5y human neuroblastoma cells . the northern blot was performed with 15 μg of total rna from human placenta and 4 μg of poly ( a ) + rna from k562 cells and from two different human placentas . the probe was the human p2y4 gene fragment amplified by pcr ( tm2 to tm7 ). on the contrary , a strong signal , corresponding to a 1 . 8 kb mrna , was found in human placenta ( fig3 ). 3 . functional expression of the new p2 receptor in 1321n1 cells after transfection of the pcdna3 - p2y4 construction in 1321n1 cells , the pool of g418 - resistant clones was tested for their functional response ( ip3 accumulation ) to atp and utp . both nucleotides were found to be agonists of the p2y4 receptor , but the response to utp was more robust . about 20 transfected clones were then isolated and tested for their response to utp . the clone presenting the highest ip accumulation factor in response to utp was selected and used in all subsequent experiments . functional characterization of the p2y 4 receptor was performed by determining the accumulation of insp 3 after 20 min incubation with the agonists in the presence of 10 mm licl . we observed that the response to utp was biphasic , with a peak reached at 30 s , followed by a more sustained stimulation of lower magnitude ( fig4 a ). with atp , only that second phase was detectable : its effect became apparent after 1 min of stimulation only and was stable for at least 20 min ( fig4 a and b ). as for utp , the stimulation by udp was biphasic , but it was slightly delayed ( fig4 a and b ). inclusion of licl had little effect on the initial peak induced by utp or udp , but it strongly enhanced the following plateau phase ( fig4 b ). the maximal effect of atp observed after a 20 min incubation represented about 27 ± 9 % of that of utp ( mean ± s . d . of ten experiments ). in order to demonstrate that atp is able to antagonize the utp response , incubations of 1321n1 cells were conducted with atp alone or in combination with utp . fig5 shows that at high concentration ( 500 μm or more ), atp was able to inhibit the effect of utp , both at 30 s and 20 min . at 30 s , the response to utp 10 μm was fully antagonized by atp 2 mm , corresponding to the fact that atp has no effect on the human p2y 4 receptor at this early time ( panel a ). at 20 min , an inhibition of 62 ± 11 % of the utp effect ( 10 μm ), corresponding to the difference between the utp and the atp effects , was observed in the presence of 2 mm atp ( mean ± s . d . of five independent experiments ) ( panels b and c ). the atp concentration - inhibition curves were shifted to the right when the utp concentration was increased , indicating the competitive nature of this inhibitory effect ( panels a and b ). on the other hand , at lower concentrations ( 30 - 300 μm ), atp enhanced the response to utp by 29 % ( range 12 - 47 %, mean of four experiments ) ( panel b ). adp , which had almost no effect per se and did not inhibit the action of utp , reproduced that enhancement : in the presence of adp ( 100 μm ), the stimulation by utp ( 10 μm ) represented 158 ± 15 % ( mean of three independent experiments ) of that by utp alone ( data not shown ). however , this potentiating effect of atp and adp was not specific : indeed the action of carbachol mediated by muscarinic receptors endogenously expressed in the 1321n1 cells ( 6 ) was also increased in the presence of these nucleotides . this observation was reproduced with cells transfected with the recombinant p2y 4 - pcdna3 plasmid or with the vector alone and was also obtained with amp and adenosine ( data not shown ). we compared the concentration - action curves of utp and udp on the insp 3 production for several clones of transfected cells . the study was made at two times ( fig6 ): 30 s and 20 min . in the set of experiments performed on clone 11 ( clone of 1321n1 transfected cells chosen for the pharmacological characterization ), utp appeared to be 10 - fold more potent than udp after a 20 min incubation and this difference was reproduced with two other clones ( fig6 ). the ec 50 values were 0 . 3 ± 0 . 1 μm and 3 . 3 ± 0 . 6 μm in clone 2 , 2 . 4 ± 0 . 1 μm and 19 . 8 ± 4 . 8 μm in clone 11 and 0 . 3 ± 0 . 1 μm and 3 . 2 ± 0 . 8 μm in clone 21 , respectively , for utp and udp ( mean ± s . d . of two independent experiments ). at 30 s of incubation , it was not possible to determine ec 50 values because the curves were clearly shifted to the right , but we can observe that the difference between the two agonists potency was even more striking ( fig6 ). several clones , including clones 2 , 11 and 21 were tested in binding studies with [ α 32 p ] utp but no increase in specific binding was observed as compared to the cells transfected with the vector alone ( data not shown ). in view of the time differences observed in fig6 the testing of a range of nucleotides was performed at two times : 30 s and 20 min . as fig7 shows , several agonists were barely or not active at 30 s ( udp , 5brutp , dutp , itp ) whereas they produced a significant effect at 20 min . full concentration - action curves were obtained at 20 min . the rank order of potency was : utp & gt ; udp = dutp & gt ; 5brutp & gt ; itp & gt ; atp ( fig8 ). the ec 50 values obtained were the following : ec 50 utp = 2 . 5 ± 0 . 6 μm , ec 50 udp = 19 . 5 ± 3 . 9 μm ( mean ± s . d . of eight independent experiments ), ec 50 dutp = 20 . 0 ± 2 . 3 μm , ec 50 5brutp = 27 . 1 ± 1 . 9 μm and ec 50 itp = 32 . 8 ± 5 . 4 μm ( mean ± s . d . of two independent experiments ). the approximative ec 50 value obtained for atp was : 43 ± 12 μm ( mean ± s . d . of five independent experiments ). the diadenosine polyphosphates also increased the insp 3 production in transfected cells with ec 50 between 3 and 7 μm ( data not shown ), but their maximal effect was only 20 - 25 % of that of utp , a value close to that of atp ( range of four independent experiments ) ( fig7 ). ump , uridine , amp , adenosine and atpγs were without any effect ( data not shown ). no specific antagonist is available for any p2y subtype . nonetheless , several non - selective antagonists such as suramin , rb2 or ppads have been tested on p 2 receptors and their relative actions on these subtypes may constitute a mean to discriminate them ( 27 ). so we tested the ability of these three antagonists to inhibit the utp response in the model of the human p2y 4 receptor . as we can see on fig9 ppads appeared to be the most active antagonist ( 73 ± 14 % inhibition ; ic 50 around 15 μm ( data not shown )), suramin was inactive , and rb - 2 produced an inhibition of 33 ± 5 % of the utp response ( mean ± s . d . of two independent experiments ). fig1 shows the mixed nature of the antagonism by ppads of the utp response : it affects both the ec 50 value and the maximal effect of utp . the ec 50 value for utp in the absence of ppads was 3 . 3 ± 0 . 6 μm and 12 . 2 ± 4 . 5 μm in the presence of 100 μm ppads ( mean ± s . d . of two independent experiments ). the effect of pertussis toxin ( 20 ng / ml , 18 hours pretreatment ) was studied at different times after utp ( 100 μm ) addition ( fig1 ). the utp response was clearly inhibited at 30 s ( 62 ± 5 % of inhibition : mean ± s . d . of two independent experiments ), whereas no significant effect was observed at 5 and 20 min . 1 . erb , l ., garrad , r ., wang , y ., quinn , t ., turner , j . t ., and weisman , g . a . 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( 1994 ) br . j . pharmacol . 113 , 614 . arg pro trp asp ala thr ala thr tyr met phe his leu ala leu ser ala his asn his trp pro phe gly thr glu ile cys lys phe val arg ile ser val his arg tyr leu gly ile cys his pro leu arg ala leu ser asn lys gly thr thr val leu cys his asp thr thr arg pro glu glu phe asp his tyr val his phe ser ser ala val met gly leu leu cys phe val pro phe his ile thr arg thr ile tyr tyr leu ala arg lys val thr arg pro leu ala ser ala asn ser cys leu asp pro val leu val ser leu pro glu asp ser ser cys arg trp ala ala thr pro