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Introduction

Te P2Y proteins belong to the amily o G-protein cou-pled receptors (GPCRs). Tey have been reported tomediate responses to extracellular nucleotides (AP/ ADP, UP/UDP) and their analogs in vivo. Eight P2Yproteins in humans, namely P2Y1, 2, 4, 6, 11, 12, 13, and14, have been cloned. Te members o this amily exert awide range o biological eects in dierent cells modu-lating cellular processes like metabolism, inammatoryresponse, and maintenance o the electrolyte balance,signal transduction, prolieration, chemotaxis, and apop-

tosis. Te dierent subtypes o P2Y receptors are associ-ated with dierent G-proteins and signaling pathways,resulting in varied physiological eects (1). Especially incase o cancer cells, P2Y receptors have been implicatedin inuencing cellular prolieration, dierentiation, andsurvival through various mechanisms at the level o the

ligand, receptor, G-protein, intracellular signaling path-ways, and surace expression (2).

A large number o P2Y receptors subtypes with hetero-geneous properties have already been characterized andmore continue to be identied. Te sequence identitybetween the members o the P2Y receptor amily can beas low as 17%. Tus, it is not a reliable indicator o purin-ergic receptor activity. Te leukotriene B4 (LB4) recep-tor has 2330% identity to other P2Y amily membersand was initially named as the P2Y7 receptor. However,urther experiments showed that this receptor was acti-vated by very low concentrations o LB4 but not even by

high concentrations o P2Y receptor agonist in culturedglioma cells (3). Similarly, the proposed P2Y9 receptorwas ound to be activated by lysophosphatidic acid (4).Furthermore, a new dual specifcity cysteinyl leukotrienereceptor has now been identied that additionally bindsto uracil nucleotides (5). Tereore, additional P2Y type

Journal of Receptors and Signal Transduction, 2011; 31(1): 7584

Address for Correspondence : Sonika Bhatnagar, Division o Biotechnology, Netaji Subhas Institute o echnology, New Delhi, 110078, India. E-mail:[email protected]

R e s e a R c h a R T I c L e

Mining human genome for novel purinergic P2Yreceptors: a sequence analysis and molecular modelingapproach

Sonika Bhatnagar, Shubhi Mishra, and Ravi Pathak

Division of Biotechnology, Netaji Subhas Institute of Technology, New Delhi, India

abtrtThe purinergic P2Y receptors are G-protein coupled receptors (GPCRs) that control many physiological

processes by mediating cellular responses to purines, pyrimidines and their analogues. They can be used aspotential therapeutic targets in a variety o disease conditions. Thereore, it is critical to identiy new mem-bers o this amily o receptors rom the human genome and characterize them or their role in health anddisease. In the present work, molecular modeling was carried out or the 21 known P2Y receptors. Bindingsite analysis was done on the basis o docking and site-directed mutagenesis data. Thus, conserved ea-tures o P2Y receptors could be ormulated. These eatures can be used to determine the purinergic natureo potential P2Y receptors in the human genome. We applied this knowledge to human genome GPCRsequences ound by sensitive sequence search techniques and identifed two orphan receptors, namelyGPR34 and GP171 that have all the necessary conserved eatures o P2Y receptors.

Kyword: P2Y receptors, G-protein coupled receptors, sequence analysis, molecular modeling, binding siteanalysis

(Received 15 August 2010; revised 28 September 2010; accepted 28 September 2010)

ISSN 1079-9893 print/ISSN 1532-4281 online 2011 Inorma Healthcare USA, Inc.

DOI: 10.3109/10799893.2010.529578 http://www.inormahealthcare.com/rst
mailto:[email protected]:[email protected]://informahealthcare.com/doi/abs/10.3109/10799893.2010.529578http://informahealthcare.com/doi/abs/10.3109/10799893.2010.529578mailto:[email protected]:[email protected]

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76 Sonika Bhatnagar et al.

receptors are expected to exist in addition to those identi-

ed. It is also possible that other already characterizedreceptors may additionally interact with and respond tonucleotides and their analogs.

In order to unambiguously identiy the sequence andstructural characteristics o P2Y receptors in the human

genome, we carried out structural modeling o all theestablished P2Y receptors. Te binding sites were ana-lyzed in line with the site-directed mutagenesis to deter-mine the residues involved in binding and specifcity. Te

mutagenesis data was also analyzed in view o the non-P2Y nature o receptors like LB4 and P2Y9. On the basiso our analysis, we identied the common eatures andpropose rules or the identifcation o new P2Y receptorsin the human genome. We applied these principles toorphan GPCR sequences identifed by sensitive sequencesearch techniques in order to predict potential purinergicP2Y receptors.

Methods

Multiple sequence alignment

Te NCBI Unigene and protein sequence database weresearched using the keyword P2Y receptor. o ensure theaccuracy o the sequence analysis, only the experimen-tally validated 21 sequences were included in the multi-ple sequence alignment (MSA) (able 1). Te sequencesobtained were aligned using MAFF (Multiple Alignment

using Fast Fourier ransorm) server, a ast and accurate

MSA program that outperorms other commonly usedsequence alignment programs (6,7). Te accuracy othe MSA was urther established by comparing thepredicted M regions o the sequences. Te M heliceswere predicted using ConPredII, a highly reliable tool orcalculating the transmembrane homology using the con-

sensus o several methods i.e., KKD, Mpred, opPredII, DAS, MAP, MEMSA 1.8, SOSUI, MHMM 2.0, andHMMOP 2.0 (8). Where the seventh helix could not bepredicted, it was interpolated by sequence similarity withother members o the group. Te nal alignment o theP2Y sequences is shown in able 2. Te unrooted tree othe sequences was constructed in Phylip (9) using theMaximum Likelihood option and is shown in Figure 1.Te reliability o the branching pattern was conrmedby generating 100 pseudoreplicates o the alignment. Teconsensus tree was then obtained.

Modeling and site analysis in the M regions

Te M regions o the 21 experimentally validated P2Ysequences were modeled in Sybyl version 6.9 (ripos Associates, Inc., St. Louis, MO, USA). Te human 2adrenergic receptor was used as template (10). Teside chains corresponding to the adrenergic receptorwere copied directly rom the crystal structure while thediering side chains were added. Te side chain posi-tions were then determined by a rotamer library searchaccording to known side chain preerences. Te helicalbundle was then minimized using the Amber Forcefeld.Te root mean square deviation o the nal minimized

structure rom the backbone atoms o the experimen-tally determined structure was 2.3 . Te site-directedmutagenesis results rom the literature were then used toidentiy and analyze the binding site. able 3 summarizesthe site-directed mutagenesis data that was used to inerthe binding site. Docking o the crystal structure o APobtained rom PDB entry 1XSC was done with the modelo the human P2Y1 receptor in agreement with the site-directed mutagenesis data. Both manual and automateddocking tools in Sybyl were used. Te residues interact-ing with docked AP in P2Y1 were also compared in themodels o the all the other P2Y receptor subtypes in orderto compare the purinergic ligand binding site.

Prole building and searching

Te MSA obtained rom the above procedures was thenused to construct a prole using the myhits server, aninteractive resource or analysis o protein sequences.Tis server (URL, http://hits.isb-sib.ch/cgi-bin/index)provides a unied resource or sequence search andanalysis (11). Te Myhits Prole search option uses theptools package to make a generalized PROSIE proleand compute new weights or each o the sequences in

able 1. List o experimentally validated P2Y sequences used astraining set or construction o prole and HMM or sequence

analysis.

No. P2Y subtype Organism Abbreviation GI number

1. P2Y1 (25) Homo sapiens P2Y1HS 4505557

2. P2Y1 (26) Gallus gallus P2Y1GG 395085

3. P2Y (27) Raja erinacea P2YRE 11993281

4. P2Y (28) Rattus norvegicus P2Y6RN 16923974

5. P2Y (29) Homo sapiens P2Y6HS 29029610

6. P2Y230 Sus scrofa P2Y2SS 54607187

7. P2Y2 (31) Rattus norvegicus P2Y2RN 8393891

8. P2Y2 (32) Mus musculus P2Y2MM 31560661




12. P2Y (36) Meleagris gallopavo P2YMG 2707256

13. P2Y (37) Xenopus laevis P2YXL 2829680




17. P2Y12 (41) Mus musculus P2Y12MM 31980659




21. GPR17 (5) Homo sapiens GPR17HS 4885301
http://hits.isb-sib.ch/cgi-bin/indexhttp://hits.isb-sib.ch/cgi-bin/index

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Mining human genome for novel purinergic P2Y receptors 77

able2.MultiplesequencealignmentoftheP2Ysequences.

Tegraybarsatthetopofthesequen

cesshowthetransmembraneregionsinP2Yreceptors.

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the MSA. Te prole obtained consisted o a matrix oposition specifc scores and gap penalties that was used to

search the database. Te score list obtained by searchingthe database with the profle is ftted to the parameters oan extreme value distribution (12). Tis generalized rep-resentation o the sequence has been ound to be muchmore sensitive or detection o remote homology.

In order to ensure the correctness o the approach,a leave one out strategy was employed. Tus, onesequence o the training set was let out while buildingthe prole. It was then determined whether the let outsequence was identied as a signicant hit in the data-base search. Tis step also served to provide an estimate

o the e-value cuto above which the hits can be treatedas signicant.

HMM building and searching

Using HMMER, a prole HMM was also built using thetraining set shown in able 1. As compared to prolemethods that use a heuristic scoring system, the prob-ability-based prole HMMs provide a consistent math-

ematical theory or the meaning and derivation o gapand insertion scores. A statistical method is used to derivethe true requency o the residue at that position o thealignment, thus making a good quality profle HMM evenwhen only 1020 sequences are used or the alignment(13). In addition, to maximize the sequence similarity,the variable N and the C-termini regions were eliminatedor the purpose o building the HMM. Te HMMER server

is available online at http://bioweb.pasteur.r/seqanal/moti/hmmer-uk.html. Te multiple local alignmentoption was used to build the model in view o the actthat the P2Y receptors have conserved M domains andvariable extracellular (EL)/intracellular loop regions. Te

hmmcalibrate option was then used or empirical deter-mination o parameters to make the search more sensi-tive. Finally, the hmmsearch option was used to searchthe database with the constructed prole HMM.

Results and Discussion

Te P2Y receptors orm three distinct subgroups

Te MSA, obtained as described above, is shown inable 2. Te unrooted tree is shown in Figure 1. It can be

able 3. Site-directed mutagenesis data used to identiy the binding

site o the human P2Y1 receptor (17).

S. No. Residue

Fold change in activity

due to mutation

Most critical residues (10050,000-old change)

1. R128 >50,000

2. R310 >50,000

3. Q307 ~207

4. K280 ~950

5. Y273a >50,000

Involved in receptor activation not ligand binding (15)Critical residues (20100-old change)

1. 221 ~100

2. 222 ~33

3. H277 ~17

4. Y136 ~17

Subcritical residues (>tenold change)

1. F131 ~7

2. H132 ~12

3. F226 ~8

P2Y2HSP2YMG P2YXL

P2Y6RN

P2Y6HS

GPCR17HSP2Y14HS

P2Y13HS

P2Y13RN

P2Y12HS

P2Y12MM

P2Y12RN

P2Y1GG

P2Y1HS

P2Y1RE

P2Y11HSP2Y4HS

ATP/UTP/UDP

Gq/o/12

ATP/ADP

Gq/s/o

Gi/o

ADP/UDP/UDP-Glu

P2Y4RN

P2Y2SS

P2Y2RN

P2Y2MM

Figure 1. Phylogenetic tree showing similarity between the various known P2Y sequences. Tree maingroups can be seen. (a) P2Y1, 11 (b) P2Y2,

4, and 6 (c) P2Y12, 13, 14, and GPCR17. Te main endogenous ligands and eector G-proteins in each group has been indicated.
http://bioweb.pasteur.fr/seqanal/motif/hmmer-uk.htmlhttp://bioweb.pasteur.fr/seqanal/motif/hmmer-uk.htmlhttp://bioweb.pasteur.fr/seqanal/motif/hmmer-uk.htmlhttp://bioweb.pasteur.fr/seqanal/motif/hmmer-uk.html

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seen that P2Y1, 11 and the receptor obtained rom Rajaerinacea orm a related group that binds to AP/ADP andits actions are mediated by Gq/11. P2Y11 additionallycouples to Gs and Go. P2Y2, 4, and 6 orms a separategroup that includes the P2Y receptors sequenced romMeleagris gallopovo andXenopus laevis. Tat is activated

by UP in addition to AP, P2Y4 is activated by uracilnucleotides in human but also by AP in rat. P2Y6 isselective or UDP. Gq/11, Go, and G12 mediate the cel-lular unctions o this group o P2Y receptors. P2Y12, 13,and 14 orm another distinct group that includes GPCR17

rom Homo sapiens. P2Y12 and 13 are activated by ADP,GPCR17 by UDP and P2Y14 by UDP-Glucose/Galactose.Te known G-proteins or this group o receptors are Giand Go (1,5).

Site-directed mutagenesis data support

ligand-receptor binding site in P2Y1 and P2Y11

Te nal structure o P2Y1 docked with AP is shownin Figure 2. In this structure, all the , , and the ter-minal phosphates are stabilized by charge interac-tions with the crucial R128(3.29) and R310(7.39) sidechains. It has been proposed that the presence o theseside chains is required to provide the counter ions orthe negatively charged phosphate groups (14). Te phosphate is additionally stabilized by the side chainso Y110(2.63), Q127(3.28), and Q307(7.36) in our model

o the P2Y1 receptor docked with AP. Accordingly, theQ307A mutation has previously been demonstratedto cause a 210-old loss o potency or agonist binding(14). Te phosphate also has charge interactions withH132(3.33) and has stabilizing interactions with the sidechain o Q127(3.28). In support o this observation, the

H132A mutation has previously been shown to cause asevenold reduction in binding potency o the agonist2MeSADP. Te AP sugar ring has interactions withH132, K280(6.55), R310(7.39), Y136(3.37), 221(5.42),222(5.43), Y306(7.35), and F276(6.51). Mutagenesis oeach o these residues except F276 is correlated with losso binding potency (15). As expected, the adenine ringoccupies a predominantly hydrophobic pocket ormedby F226(5.47), 221, 222, M225(5.46), F276, H277(6.52),Y136, Y273(6.48), F131(3.32), and H132. A schematic othe adenine ring surrounded by a number o hydrophobicresidues on both sides is shown in Figure 3. In our model

o the P2Y1 receptor, the S314(7.43) residue did not makeany contact with the docked AP. It was also observedthat the corresponding positions did not have serine resi-dues in P2Y 11, 12, 13, 14, and GPCR17. However, it hasbeen reported that the S314A mutant receptor does notexhibit any activity (16). Tis residue may be involved insignal transduction without any eect on ligand binding.Te residues interacting with AP in P2Y1 were compared

in all the P2Y receptors to determine the set o essentialresidues and interactions. Te comparison o the binding

H132

ATP

TMVTMVI TMVII

TMIV TMIIITMII

TMI

R128

Q127

L107

Y110

Q307

F131

R310

Y306

L135

Y273

K280

H277

F226

T222

S218

M225

T221

Y136

Figure 2. Te AP-binding pocket o the human P2Y1 receptor is shown. Te side chains rom each o the M helices has been color coded as

shown and labeled in the inset. Te AP in its nal docked position is shown as black sticks. Te inset shows a side view o the seven M helices

o the human P2Y1 receptor. Te docked AP has been rendered as black sticks.

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site residues in the dierent groups o P2Y receptors is

shown in able 4.Te binding site o P2Y11 is similar to that o P2Y1.Te residues R106(3.29) and R307(7.39) o P2Y11 (cor-responding to R128 and R310, respectively in P2Y1) arecritically required or the receptor unction. Interaction with the F109(3.32) and R268(6.55) (corresponding toF131 and K280, respectively in P2Y1) is also supportedby mutagenesis experiments resulting in loss o agonistpotency (17). Te position corresponding to H132 oP2Y1 is occupied by the smaller , making the bindingpocket more spacious. Tis is in correspondence with therecent nding that the larger NAD+ is also an agonist atthe human P2Y11 receptor.

A critical tyrosine hinders AP binding at the P2Y2, 4,

and 6 receptor binding site

Te main dierence in the binding site o the P2Y2receptor as compared to P2Y1 can be attributed to Y114/116(3.33) (H132 in P2Y1). Te H132 residue points inward

and upward in the AP-binding site (Figure 2). A subtlechange o H to Y causes it to hinder the AP penetrationinto the pocket as compared to P2Y1. Te presence oK289(7.36) (in place o Q307 o P2Y1) as a -phosphateinteracting residue may help to tether the ligand in thisposition. Additionally, the shape o the adenine pocket

in P2Y2 is changed due to the presence o M202(5.42)(221 in P2Y1) and L203(5.43) (222 in P2Y1). ogether,these three actors may account or the act that P2Y1group o receptors bind to adenine nucleotides, whereasP2Y2 and P2Y4 additionally bind to the smaller uridinenucleotides.

In the P2Y6 receptor binding site, the residue corre-sponding to H132 in P2Y1 is Y107(3.33) as in the abovetwo cases. Te residue corresponding to Q307 is againK284(7.36). Te residues corresponding to the 221 isreplaced by L196(5.42) whereas the other 222(5.43)

position is conserved. P2Y6 is also activated by uridinenucleotides, indicating that the residues as discussedabove are the main determinants o ligand specicity.Mutagenesis data is not available or any o these recep-tors at present.

A diferent set o Arg and Lys residues are responsible

or phosphate group binding at the P2Y12, 13, and 14

and GPCR17 receptor binding site

In this group, uncharged groups like S/A/G and L/S sub-stitute the residues corresponding to the critical R128 and

R310 residues o P2Y1, respectively. However, modelingstudies indicate that K80(2.60) and K280 in P2Y12 (cor-responding to L107(2.60) and Y306 in the binding siteo P2Y1) can compensate or the absence o positivelycharged residues. As the charged groups in the ligandalign at avorable positions with respect to these side

chains, the position o the ligand in the binding site isexpected to be dierent as compared to P2Y1. Te pres-ence o three additional H bonding residues, namelyN159, N164, and N191 in place o I186(4.60), S218(5.39),and G191(4.64) o P2Y1 may provide extra stabilizinginteractions or the phosphate groups. Te same changesare ound in the active site o P2Y13 also. Te reason orbinding mainly to diphosphates in this group is alsoapparent rom the analysis o the binding site. Te residue

corresponding to Q307 o P2Y1 is E281(7.36). As Q307 isclosely interacting with and has hydrogen bonds to the-phosphate, the negatively charged E281 o P2Y12 in thisposition is likely to cause considerable charge repulsion

that makes the triphosphate group binding unavorable.It may be possible that the ADP binding at this receptoris stabilized by E281 through Mg2+ ion as proposed or theP2Y1 receptor (18).

Te last member o this group, namely GPCR17 alsolacks the K/R residues at the positions corresponding toR128 and R310 o P2Y1Hs. Te counterions or the phos-phate group in this case are likely to be provided by theR115(7.36) (corresponding to Q307 o P2Y1). Te pocketor glucose moiety o the UDP-glucose ligand o P2Y14lies toward the EL loops and was not modeled.

Critical positively charged residues are required orbinding and activation in diferent P2Y subtypes

A study o the binding site indicates that there are sev-eral positions o the M helices at which the presenceo positively charged residues is avorable or interactionwith the phosphate moieties and sugar ring in AP. Tesepositions include:

Te position corresponding to R128 (MIII) o P2Y1.Tis residue is conserved in P2Y 1, 2, 4, 6, and 11receptors.

H277*F276

N N

N

NN

N N

Adenine

N

N

N

N

N

NNN

N

N

N

N

O

O

O

O

OO

O

O

OS

O

O

O

O

O

M225

T222*

T221*

F226* H132*

F131*

Y273*

Y136*

Figure 3. Schematic o the residues surrounding the adenine-binding

pocket o the P2Y1 receptor.*Residues or which mutagenesis experi-

ments show a loss o binding potency or agonist.

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able4.Comparisonoftheresidues/Mregionsoftheligand-bindingsiteofP2Y

1withthecorrespondingresiduesinotherP2Yreceptors.

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Te residue corresponding to K280 (MVI) o P2Y1.It is K/R in all the known P2Y receptors.Te residue corresponding to Y306 (7.35) o P2Y1. Itis replaced by K in P2Y12, 13, 14 and N in GPCR17.Te position corresponding to L107(2.60) o P2Y1. Asshown in able 4, it is replaced by K in P2Y12, 13, and

14 and by R in GPCR17.Te residue corresponding to G191 (MIV) in P2Y1.Tis is replaced by R in P2Y rom R. erinacea and Hin P2Y11.Te residue corresponding to Q307 (MVII) inP2Y1. It is present as K in the P2Y2, 4, 6 group andin GPCR17.Te residue corresponding to R310 in P2Y1. Itremains R in both the P2Y1, 11 as well as the P2Y2,4, 6 group.

Common conserved sequence and structural eatures

o all P2Y receptors

Apart rom the presence o K/R residues at avorablepositions, other sequence eatures were also ound tobe conserved and important or activity within the P2Ygroup. On the basis o analysis o sequence conservationand site-directed mutagenesis data, it was ound that theollowing sequence eatures are conserved in all the P2Ysequences.

Cysteine residues corresponding to positions 42 o1.the N-terminus and position 296 in EL3 as well asresidues 124 in the EL1 and 202 in the EL2 o P2Y1.

Tough these residues are present in the ELs and donot directly orm part o the ligand-binding site, therst two orm a disulphide bridge and are requiredor surace expression o the P2Y receptors. Te latterpair o cysteine residues are required or both suraceexpression and activity. It has been proposed that thetwo-disulde bridges help to constrain and stabilizethe structure (19). In the P2Y12 receptor, it has beendemonstrated that the cysteine residues 17 and 270(corresponding to the cysteine 42 and 296 in P2Y1)do not orm a disulfde bond. However, these cysteine

residues are proposed to be involved in interaction

o the receptor with Tiol reagents, thus leading to itsinactivation (20). Te our cyteines are conserved inall known P2Y receptors and may thus be taken to beessential or their expression and activity.

Te presence o a HXXK/R moti in MVI is common2.to all P2Y receptors. In MVII, a moti has previouslybeen detected (16). We ound that this second motiin MVII is QXXR or the P2Y1, 11 group, KXXR orthe P2Y2, 4, 6 group, KEXXL or the receptors P2Y12,13, 14. However, the moti is not present either in theGPCR17 or in CLR1, both o which are purinergic

P2Y receptors. Tereore, it may be concluded thatthe frst moti in MVI is essential or activity whereasthe second one in MVII is not.

Analysis o the presence o avorable positively3.charged residues showed that at least three suchgroups were required in each o the P2Y receptorgroups. It is proposed that in the P2Y1, 11 as wellas the P2Y2, 4, 6 group, these include the residuescorresponding to K125(EL1), R128 and R310 oP2Y1. For P2Y12, 13, and 14, the positively chargedresidues or phosphate and sugar interaction are theresidues corresponding to K80(2.60), with additional

charge groups being provided by the residues cor-responding to R256(6.55) and K280(7.35) o P2Y12.Tese charged residues correspond to L107, K280,and Y306, respectively in P2Y1. In GPCR17, theseinclude R283(6.55), R115(2.60), and R308(7.36)(corresponding to residues K280, L107, and Q307 o

P2Y1, respectively).Te sugar ring also requires stabilization by a polar4.group. Tis is provided by the K/R residues corre-sponding to K280 o P2Y1. Tis position is completely

conserved and is always K/R in all the known P2Yreceptors. Other stabilizing inuences may also begained by interaction o the ring with residues H132,Y136, and Y306 o P2Y1. Te residues correspondingto these in other P2Y receptors are also hydrogenbonding.

A number o hydrophobic interactions are made by5.the nucleotide base o the ligand with the side chains

o dierent residues o P2Y1 receptor as summarizedin Figure 3. O these, residues Y273 and F226 o P2Y1are expected to be most critical as indicated bysequence conservation in all the other P2Y receptorsas well as by mutagenesis studies in P2Y1.

Te two residues, E209 (EL2) and R287 (EL3) o P2Y1receptor have previously been proposed to be impor-tant or ligand binding and activity. Mutation o eithero these residues in P2Y1 is responsible or more than1,000-old loss o potency as compared to the wild-typereceptor. Although, the two have been proposed to beinvolved in H bond in P2Y1, the corresponding position

in other P2Y receptors is occupied by residues that can-not be involved in H bond ormation. Tereore, it is notlikely to be a universal eature o P2Y receptors. However,

the R287 residue is always occupied by R/Q and is Y incase o the GPCR17. It has been proposed to be involvedin the ormation o an additional meta-binding site orbinding o ligand within the EL region (21,22). Tereore,while the presence o R287 is signicant in view o itspositional conservation, it may not be always involvedin hydrogen bond with residue corresponding to E209o P2Y1.

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Application o identied conserved eatures to orphan

GPCRs in the human genome

Potential P2Y GPCR sequences were identied in thehuman genome by sequence profle and HMM methods.Te conserved eatures o P2Y receptors as described

above were applied to these GPCR sequences. Using thismethod, two orphan human GPCR sequences could beidentied as potential P2Y receptors. Tese are GPR34(Uniprot ID: Q9UPC5) and GP171 (Uniprot ID: O14626),which are known to be close to P2Y12, 13, 14 receptors(23,24).

Conclusion

In the past, it has been difcult to determine the P2Ynature o purinergic-like receptors on the basis osequence analysis alone. Also, with the discovery o

GPCRs that bind AP in addition to other ligands, the so-called dual receptors, the possibility o other GPCRs withpurinergic ligand-binding properties has become evi-dent. Tereore, a method or unambiguously identiyingP2Y receptors is required. For this, the essential motis,sequence, structural eatures o the P2Y group need tobe elucidated. We have integrated multiple methods osequence analysis and molecular modeling in view o theexperimentally determined mutagenesis data to postulate

rules or the unambiguous detection o the P2Y natureo similar GPCRs. Using the rules as outlined above, itis possible to exclude the non-P2Y sequences with highsimilarity to the P2Y receptors like P2Y9, oxoglutaratereceptor, etc. Tus, we could identiy two novel potentialP2Y receptors in the human genome.

Declaration of interest

S.B. would like to acknowledge the nancial support orpurchase o Sybyl sotware by the Department o Scienceand echnology, New Delhi, India.

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