Patent Application: US-7717398-A

Abstract:
this invention provides a receptor having a preference for pyrimidine nucleotides preferably uridine triphopshate over purine nucleotides . a receptor having a preference for pyrimidine nucleotides over purine nucleotides means a receptor for which pyrimidine nucleotides and purine nucleotides are not equally active and equipotent . this means that the receptor according to the invention in presence of these agonists presents a functional response , diacylglycerol , or calcium ions ) to lower concentration of pyrimidine nucleotides , preferably uridine triphopshate , than to purine nucleotides or a more important functional response to similar concentration of pyrimidine nucleotide than to purine nucleotide .

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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