Identification and characterization of a cell-surface ... · with homology to the P2Y family of...

Identification and characterization of a cell-surface receptor, P2Y15, for AMP

and adenosine

Hisayo Inbe, Shinichi Watanabe, Miwa Miyawaki, Eri Tanabe, and Jeffrey A. Encinas*

Bayer Yakuhin, Ltd., Research Center Kyoto, 6-5-1-3 Kunimidai, Kizu-cho, Soraku-gun, Kyoto

619-0216, Japan

* To whom correspondence should be addressed:

Jeffrey A. Encinas

Bayer Yakuhin, Ltd., Research Center Kyoto

6-5-1-3 Kunimidai, Kizu-cho, Soraku-gun, Kyoto 619-0216, Japan

Tel.: +81-774-75-2468; Fax: +81-774-75-2506

E-mail: [email protected]

Running title: Identification of a Receptor for AMP and Adenosine

Keywords: AMP, adenosine, P2Y15, G protein-coupled receptor

JBC Papers in Press. Published on March 4, 2004 as Manuscript M400360200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on March 19, 2020

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Summary

AMP and adenosine are found in all cell types and can be released by cells or created extracellularly

from the breakdown of ATP and ADP. We have identified an orphan G protein-coupled receptor

with homology to the P2Y family of nucleotide receptors that can respond to both AMP and

adenosine. Based on its ability to functionally bind the nucleotide AMP, we have named it P2Y15.

Upon stimulation, P2Y15 induces both Ca2+ mobilization and cyclic AMP generation, suggesting

coupling to at least two different G proteins. It is highly expressed in mast cells and is found

predominantly in the tissues of the respiratory tract and kidneys, which are known to be affected by

AMP, adenosine, and adenosine antagonists. Until now, AMP’s effects have been thought to depend

on its dephosphorylation to adenosine, but we demonstrate here that P2Y15 is a bona fide AMP

receptor by showing that it binds [32P]-AMP. Since AMP and adenosine have bronchoconstrictive

effects that can be inhibited by theophylline, we tested whether theophylline and other adenosine

receptor antagonists can block P2Y15. We found inhibition at a theophylline concentration well

within the therapeutic dose range, indicating that P2Y15 may be a clinically important target of this

drug.

Abbreviations used in this paper: AMP, adenosine 5’-monophosphate; ATP, adenosine 5’-

triphosphate; ADP, adenosine 5’-diphosphate; GPCR, G protein-coupled receptor; RT-PCR, reverse

transcription-polymerase chain reaction; ADA, adenosine deaminase; IBMX, 3-isobutyl-1-

methylxanthine; 8-SPT, 8-(p-sulfophenyl)theophylline; 8-PT, 8-phenyltheophylline; DPCPX, 8-

cyclopentyl-1,3-dipropylxanthine; CSC, 8-(3-chlorostyryl)caffeine; NECA, 5’-(N-

ethylcaboxamido)adenosine; Chloro-IB-MECA, 2-chloro-N6-(3-iodobenzyl)-adenosine-5’-N-

methyluronamide.


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Introduction

Adenosine can mediate diverse physiological effects including bronchoconstriction, inhibition of

platelet aggregation, inhibition of lipolysis, induction of sedation, vasodilation, suppression of

cardiac rate and contractility, and stimulation of gluconeogenesis. To date, five cell-surface

receptors have been identified for adenosine, namely, adenosine receptors A1, A2A, A2B, and A3

(collectively referred to as P1 receptors), and the growth hormone secretagogue receptor GHSR

(1,2). AMP has a similarly diverse repertoire of effects (3-6), including bronchoconstriction,

stimulation of DNA synthesis, mitogenesis, and stimulation of chloride secretion, but no receptor

for AMP has previously been reported. Numerous cell types can release adenosine, including mast

cells (7), kidney brush border cells (8) and cardiac cells (9), while AMP can be released by such cell

types as activated platelets (10), neutrophils (4), and eosinophils (11). AMP can also be generated

extracellularly from the hydrolysis of ATP and ADP by ecto-ATPases (12) and ecto-ATP

diphosphohydrolases (13), and can be further dephosphorylated by ecto 5’-nucleotidases to produce

adenosine (14). A number of widely-used drugs have been developed, such as theophylline (15) and

cromolyn (16), that can modulate what are thought to be the effects of adenosine in diseases such as

asthma, but their mechanisms of action are not yet fully understood.

Other extracellular nucleotides similarly induce a wide variety of responses in many cell types,

including muscle contraction and relaxation, vasodilation, neurotransmission, platelet aggregation,

ion transport regulation, and cell growth. The effects are exerted mainly through two types of cell

surface molecules: P2Y type G protein-coupled receptors (GPCRs), and P2X type ligand-gated ion

channels. Nine nucleotide-stimulated P2Y type GPCRs have been characterized to date in humans:

P2Y1, P2Y11, P2Y12, and P2Y13 which are activated by the adenine nucleotides ATP or ADP;

P2Y4, P2Y6, P2Y14,and CYSLT1 which are activated by the uridine nucleotides UTP or UDP (or

in the case of P2Y14, UDP-glucose); and P2Y2 which is activated by both adenine and uridine


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nucleotides (17-20). None of these receptors has been shown to be able to bind adenosine or AMP.

Here we report on the characterization of a GPCR with close homology to the P2Y receptors that

can bind and respond to both AMP and adenosine.

Experimental Procedures

P2Y15 cDNA Cloning. Protein sequences of known P2Y receptors were used to search for

homologs in the Genbank database of the National Center for Biotechnology Information

(http://www.ncbi.nlm.nih.gov) using the program tblastn. The search identified an intronless

genomic sequence subsequently also found by others and designated in Genbank as the orphan

receptor GPR80 (21). To clone the gene, human genomic DNA was used as template, and PCR was

performed using primers 5’-GCCAAACTGAACTCTCTTGTTTTCTTGC-3’ and 5’-

GCCCTGGCTTTGGCACATGATTAC-3’ and a blend of HotStarTaq (Qiagen, Hilden, Germany)

and Pfu Turbo (Stratagene, La Jolla, CA) polymerases. PCR products were cloned into pCRII-

TOPO (Invitrogen, Carlsbad, CA), cycle-sequenced with an ABI Prism Dye Terminator Cycle

Sequencing Reaction Kit (Applied Biosystems, Foster City, CA), and analyzed on an ABI Prism

377 sequencing system (Applied Biosystems). For functional studies, the cDNA was subcloned into

a pDisplay vector (Invitrogen) to append an N-terminal HA epitope and Igκ signal sequence.

Expression profiling. 25 µg of total RNA from the following were used as template in reactions to

synthesize first-strand cDNA for expression profiling: Human Total RNA Panel I-V (Clontech

Laboratories, Palo Alto, CA), normal human lung primary cell lines (BioWhittaker Clonetics,

Walkersville, MD), several common cell lines (ATCC, Washington, DC), and various cells purified

from peripheral blood. First-strand cDNA was synthesized using oligo (dT) (Nippon Gene Research

Laboratories, Sendai, Japan) and the Superscript™ First-Strand Synthesis System for RT-PCR (Life


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Technologies, Rockville, MD) according to the manufacturer’s protocol. For these samples, 1/1250th

of the synthesized first-strand cDNA was subsequently used as template for quantitative PCR.

Additional samples were purchased as presynthesized cDNAs (Human Immune System MTC Panel

and Human Blood Fractions MTC Panel, Clontech Laboratories), and for these, 10 ng of cDNA was

used as template for quantitative PCR.

Quantitative PCR was performed in a LightCycler (Roche Molecular Biochemicals, Indianapolis,

IN) with oligonucleotide primers 5’-TTCGGATCGAATCTCGCCTGCT-3’ and 5’-

TGCTTGCTCAAGGTTCCCGCTTA-3’ in the presence of the DNA-binding fluorescent dye

SYBR Green I. Results were then converted into copy numbers of the gene transcript per ng of

template cDNA by fitting to a standard curve. The standard curve was derived by simultaneously

performing the quantitative PCR reaction on PCR products of known concentrations amplified

beforehand from the target gene.

To correct for differences in mRNA transcription levels per cell in the various tissue types, a

normalization procedure was performed using similarly calculated expression levels of five

different housekeeping genes: GAPDH, hypoxanthine guanine phophoribosyl transferase, beta-actin,

porphobilinogen deaminase, and β2M. Expression levels of the five housekeeping genes in all tissue

samples were measured in three independent reactions per gene using the LightCycler and a

constant amount (25 µg) of starting RNA.

Expression levels were also measured using CodeLinkTM microarrays (Amersham Biosciences,

Buckinghamshire, England). Total RNA was prepared from human umbilical cord blood-derived

mast cells (kindly provided by Professor H. Nagai, Department of Pharmacology, Gifu

Pharmaceutical University, Gifu, Japan; prepared as described in (22)) and purified leukocyte

fractions, and then used to synthesize biotin-labeled cRNA using the Amersham cRNA synthesis kit.

cRNA yield was quantified by measuring absorbance at 260 nm, and then the cRNA was

fragmented in 40 mM Tris–acetate (TrisOAc) pH 7.9, 100 mM KOAc and 31.5 mM MgOAc, at


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94°C for 20 min. Ten micrograms of fragmented cRNA from each sample was used for

hybridization to a CodeLinkTM UniSet 20K Human Expression Bioarray chip (Amersham

Biosciences). cRNAs bound to the microarrays were stained with streptavidin-Cy5 and the

processed slides were scanned with an Axon GenePix 4000B Scanner. Images for each slide were

analyzed using the CodeLinkTM Expression Analysis Software (Amersham Biosciences).

Determination of ligand specificity. HA-tagged P2Y15 was transfected into HEK293 cells (ATCC)

using Lipofectamine (Invitrogen). Expression on the cell surface was verified by staining cells with

phycoerythrin-labeled anti-HA antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and

measuring fluorescence on a FACSort (Becton-Dickinson, Franklin Lakes, NJ). Stably transfected

clones were generated by selection in G418 (500 µg/ml) and reconfirmed for cell-surface

expression of the P2Y15 protein. Ligand screening was performed in a Ca2+ mobilization assay as

follows: Stably transfected P2Y15 GPCR-expressing cells were seeded into 96-well plates and

incubated overnight at 37 °C. The culture medium was aspirated and replaced with 100 µl of

loading buffer consisting of 0.1% BSA, 20 mM HEPES, 1 mM probenecid, 0.01% pluronic F127,

and 1 µM Fluo-3-AM (Molecular Probes, Eugene, OR) in HBSS, and incubated for 1 hour at room

temperature. The cells were then washed gently 3 times with wash buffer consisting of 0.1% BSA,

20 mM HEPES, and 1 mM probenecid in HBSS. The washed cells were placed in an FDSS6000

functional drug screening system (Hamamatsu Photonics, Hamamatsu, Japan) and changes in

cellular fluorescence were measured after adding serial dilutions of potential ligands. A panel of

about 130 potential ligands for testing was assembled by selecting known ligands of the GPCRs

most closely related to P2Y15 GPCR as well as several naturally occurring chemical relatives of the

ligands. The panel included various bioactive lipids, eicosanoids, peptides, cannabinoids,

chemokines, nucleosides, nucleotides and chemically related substances which were generally


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purchased from either Sigma (St. Louis, MO) or R&D Systems (Minneapolis, MN).

Ligand confirmation analyses were performed by repeating the Ca2+ mobilization experiments

using the identified ligands to stimulate P2Y15 transfectants, identically constructed P2Y8

transfectants, and non-transfected HEK293 cells, all of which had been cultured for two hours with

or without 1 µM pertussis toxin. Antagonist assays for inhibition of calcium responses to AMP and

adenosine were performed essentially as above except that serial dilutions of antagonist compounds

were added five minutes prior to the addition of ligand. Agonist assays for stimulation of calcium

responses were performed in the same manner as the ligand screen.

AMP conversion assays. Transfected and nontransfected cells in DMEM medium were seeded at

105 cells per well into 96-well plates and incubated for 3 hours. The medium was then exchanged

with fresh medium and 1.85 µM [3H]-AMP (Amersham) was added to the wells. After incubation

for 5 or 60 min, 6 µl of the medium, together with AMP and adenosine standards, were applied to

thin layer chromatography sheets, separated with isobutyl alcohol/isoamyl alcohol/2-

ethoxyethanol/ammonia/H2O (9:6:18:9:15) as solvent, and visualized under UV light as described

by Yegutkin et al. (23). The spots corresponding to AMP and adenosine were cut from the sheets

and the amounts of each were quantified by scintillation counting.

Receptor binding assays. 105 cells per well in 96-well plates were washed twice for 1 h with

DMEM medium. Wheatgerm agglutinin SPA beads (Amersham) were then added at 1 mg/well,

followed 1 h later by the addition of increasing concentrations of [3H]-Adenosine or [3H]-AMP

(Amersham), in a constant volume of HBS (10 mM Hepes, 130 mM NaCl, 5 mM KCl, 1 mM

MgCl2, 1 mM CaCl2 and 1 g/l glucose (6 µl/well)). After incubating at 4 °C for 16 h, the plates were

centrifuged for 10 min at 1500 rpm and then scintillation measured on a TopCount automated


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scintillation counter. Non-specific binding measurements and competitive binding experiments were

carried out under the same conditions but with either an excess of cold ligand (2.5 mM) or

increasing concentrations of cold ligand, respectively. Adenosine binding in competitive binding

experiments was measured after incubation for 1 hr at 4 °C. Binding of [32P]-AMP was carried out

under the same conditions except that instead of using SPA beads, at the end of the incubation, cells

were washed three times by vacuum filtration and 100 µl scintillation fluid was added to the wells.

Kd values were determined by non-linear regression using the program Prism (Graph Pad Software,

San Diego, CA).

Cyclic AMP production assay. Cyclic AMP production after stimulation of cells was measured with

the Tropix cAMP-screen (Applied Biosystems) according to the manufacturer’s protocol. Briefly,

stable transfectants and control cells (1 x 105 cells/well) were cultured for two hours with or without

1 mM pertussis toxin, then treated for 30 min with 10 mM forskolin and serial dilutions of AMP or

adenosine. The cells were then lysed and the cAMP produced was measured by a cAMP-specific

ELISA. Concentrations of cAMP produced were calculated by comparing against cAMP standards

measured simultaneously. The effect of adenosine deaminase (ADA) on ligand-stimulated cyclic

AMP production was measured essentially as above except that pertussis toxin was excluded and

cells were treated for 30 min with 10 mM forskolin prior to the addition of 25 mM adenosine or

100 mM AMP. Serial dilutions of ADA (Roche Diagnostics, Tokyo, Japan) were added to the

cultures 10 minutes prior to the addition of ligand.

Results

Cloning and Sequencing. In an effort to find new receptors for extracellular nucleosides and

nucleotides, we searched for homologs of known P2Y nucleotide receptors in the Genbank database


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of the National Center for Biotechnology Information using the program tblastn. P2Y receptors are

receptors for diphosphate and triphosphate adenine and uridine nucleotides, though none respond to

AMP or adenosine. Our search identified an intronless genomic sequence subsequently also found

by others and designated variously as the orphan receptor GPR80 (21) or GPR99 (24). We are now

renaming it P2Y15 as a new member of the P2Y family. A putative mouse ortholog of the gene has

recently appeared in Genbank under accession number XP_139267. We subsequently found the rat

ortholog by using the mouse protein sequence in a tblastn query against rat genome sequences. An

alignment of the human, mouse, and rat protein sequences is shown in figure 1.

The conceptually translated protein product of the human P2Y15 gene shows 36% identity over

its full length with the nucleotide receptor P2Y1, increasing to an overall sequence similarity of

58% when amino acids with related physicochemical properties are included. Homology of the

protein sequence with other P2Y nucleotide receptors P2Y2, P2Y4, P2Y6, and P2Y11 likewise

shows an overall identity ranging from 25 to 35% and a similarity ranging from 43 to 57%. A

similar, though slightly lower, level of homology is seen among the mouse and rat orthologs

(excluding P2Y11, which has not yet been found in rodents). The human gene transcript encodes a

polypeptide of 337 amino acids with a calculated molecular mass of 38.3 kD. A phylogenetic

analysis comparing the protein sequence with other GPCRs places the molecule among a cluster of

other P2Y receptors, distant from the known receptors for adenosine (figure 2). The gene sequence

is found on the genomic contig NT_009952 which has been localized to human chromosome

13q32.

Tissue Distribution of P2Y15. As a first step to investigating the the function of P2Y15, we

examined the distribution of P2Y15 messenger RNA expression in several different human tissues,

cell types, and commonly used cell lines. We designed oligonucleotide primers near the 3’ end of

the coding region that could specifically amplify P2Y15 cDNA and used these in a quantitative

reverse transcription-polymerase chain reaction (RT-PCR) analysis to measure relative transcript


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levels. Among the tissues tested, trachea, salivary glands, kidney, fetal brain and lung showed the

highest expression levels (figure 3a). The high expression in the respiratory tract prompted us to

look in depth at the cell types within the trachea and lung that might be responsible for the

predominant expression there. Little or no expression, however, could be detected in any of the

tested primary cell populations or transformed cell lines derived from lung tissues or from immune

cell subtypes (figure 3b).

We therefore investigated the possibility that the gene is expressed in a minor population of cells in

the respiratory tract such as mast cells or eosinophils. Because mast cells and eosinophils typically yield

only small amounts of mRNA, we analyzed the expression of P2Y15 in these cells by microarray

analysis which allows the analysis of more genes than quantitative RT-PCR and provides multiple

controls to verify the quality of the sample preparations. Analysis of gene expression in CD4 and CD8 T

cells, tonsil B cells, neutrophils, eosinophils, and mast cells showed that the P2Y15 gene is expressed

specifically and at very high levels in mast cells (figure 3b). Indeed, the high level of P2Y15 expression

in mast cells places it among the top 1% of the approximately 20,000 different gene probes included in

the microarray. Similar specificity and high level expression in these cell types was not seen with any of

the other P1 adenosine receptors (figure 3c) or P2 nucleotide receptors (data not shown).

Identification of AMP and Adenosine as Functional Ligands for P2Y15. To identify the ligand of

P2Y15, we generated stable transfectants with HEK293 cells and then tested for calcium mobilization in

response to a panel of ligands. Among the potential ligands tested, only AMP and adenosine were able to

induce a response in the transfectants while not inducing a similar response in either nontransfected

HEK293 cells or HEK293 cells stably transfected with the control orphan GPCR P2Y8 in an identical

vector construct. We detected a calcium response with an EC50 of 920 nM for AMP and 670 nM for

adenosine (figure 4a). The calcium response to either ligand was not significantly affected by pertussis

toxin (data not shown). Both stable transfectants and nontransfected cells mobilized calcium in response

to ATP, ADP, and UTP, consistent with previous reports of HEK293 endogenously expressing P2Y1 and


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P2Y2 receptors (25). Further analysis by RT-PCR showed that the nucleotide receptors P2Y4, P2Y12,

and P2Y13 and the adenosine receptors A2A and A2B (previously reported in (26)) are also expressed in

HEK293 cells (data not shown). Despite the endogenous expression of the adenosine receptors, however,

calcium mobilization responses to adenosine in nontransfected cells could only be detected at very high

adenosine concentrations, and showed only a very weak response (figure 4a).

To determine the effect of P2Y15 stimulation on adenylate cyclase activity, we measured cyclic

AMP accumulation in our P2Y15-HEK293 stable transfectants in response to AMP and adenosine.

Stimulation with either ligand alone gave only minimal responses barely above the detection limit.

In the presence of 10 µM forskolin, however, both AMP and adenosine induced the generation of

cyclic AMP in a dose dependent manner, with an EC50 of 214 nM for AMP and 327 nM for

adenosine (figure 4b). Nontransfected HEK293 cells similarly generated cyclic AMP in response to

adenosine, likely due to the stimulation of endogenously expressed adenosine receptors, but did not

respond strongly to AMP. The production of cyclic AMP in response to either ligand was not

affected by pretreatment of the cells for two hours with 1 µM pertussis toxin (data not shown),

indicating that P2Y15 does not couple with an adenylate cyclase-inhibiting G protein. The

responsiveness of the P2Y15 transfectants to AMP in the cyclic AMP assay did not appear to be due

to an increased conversion rate of AMP to adenosine, since transfected and non-transfected cells

showed similarly low rates of endogenous nucleotidase activity (figure 4c).

Saturation binding analysis of the ligands to the P2Y15 receptor in stable transfectants gave Kd

values of 12.0 µM for [2-3H]-adenosine (figure 5a) and 18.6 µM for [2-3H]-AMP (data not shown).

Since AMP can be dephosphorylated to adenosine by ectonucleotidases, we repeated the binding

analysis with adenosine 5’-[32P] monophosphate to confirm that the binding being measured was

AMP and not adenosine. This resulted in a similar binding curve with a Kd of 18.8 µM (figure 5b),

indicating that AMP itself, and not a breakdown product, was binding to the receptor. We then

performed competitive binding assays to determine whether one ligand could antagonize the


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binding of the other ligand to the P2Y15 transfectants. While unlabeled AMP was able to block a

large proportion of the binding of 10 µM [2-3H]-adenosine to transfectants (Ki = 32.0 µM), it did

not block the binding as completely as unlabled adenosine and had little effect in blocking 3H-

adenosine binding to nontransfected HEK293 cells (figure 5c). On the other hand, unlabled

adenosine was able to block the binding of 10 µM [2-3H]-AMP to transfectants (Ki = 39.8 µM) with

a potency similar to that of unlabled AMP (figure 5d).While these results provide evidence that both

AMP and adenosine bind to P2Y15, the results also demonstrate a lack of specific AMP binding

sites on the nontransfected cells since neither AMP nor adenosine could antagonize the binding of

[2-3H]-AMP to nontransfected HEK293 cells beyond the background level.

To further clarify whether AMP itself, without its conversion to adenosine, is able to induce a

response in P2Y15 transfectants, we measured AMP- and adenosine-induced cyclic AMP

production in the presence of adenosine deaminase (ADA), an enzyme that breaks down adenosine

to inosine. As expected, ADA inhibited the adenosine-induced response in a dose-dependent manner,

but showed little if any effect against the AMP-induced response, indicating that adenosine is not a

mediator of the AMP response (figure 6).

Characterization of Antagonists and Agonists of the P2Y15 Receptor. To test whether any of the

known antagonists or agonists of adenosine receptors could antagonize P2Y15, we performed

calcium mobilization assays in the presence of varying concentrations of such compounds.

Antagonists tested were the non-selective adenosine receptor antagonists theophylline, caffeine, 3-

isobutyl-1 methylxanthine (IBMX), and 8-(p-sulfophenyl)theophylline (8-SPT); selective A1

receptor antagonists 8-phenyltheophylline (8-PT) and 8-cyclopentyl-1,3-dipropylxanthine

(DPCPX); the selective A2A receptor antagonist 8-(3-chlorostyryl)caffeine (CSC); and the selective

A2B receptor antagonists enprofylline and alloxazine. With the exception of alloxazine and DPCPX

which were agonistic in both transfected and non-transfected cells, all of these compounds were

able to block the calcium mobilization induced by AMP and adenosine, with Ki values for blocking


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AMP ranging from 200 nM for 8-SPT to 3,309 nM for caffeine (figure 7a, table 1) and Ki values for

blocking adenosine ranging from 962 nM for 8-theophylline to 64,060 nM for enprofylline (figure

7b, table 1). Since many of these compounds can also act as phosphodiesterase inhibitors and cause

an increase in cyclic AMP levels that can potentially inhibit Ca2+ mobilization, the compounds were

also tested for their ability to inhibit ADP-induced Ca2+ mobilization in the same cells. None of the

compounds, however, had significant inhibitory effects at concentrations less than 1 mM (data not

shown). Adenosine receptor agonists tested were the non-selective agonist 5’-(N-

ethylcaboxamido)adenosine (NECA); the selective A1 receptor agonist N6-cyclopentyladenosine;

the selective A2A receptor agonist CGS-21680 hydrochloride; and the selective A3 receptor agonist

2-chloro-N6-(3-iodobenzyl)-adenosine-5’-N-methyluronamide (Chloro-IB-MECA). Among these

componds, only NECA and N6-cyclopentyladenosine showed measurable agonist effects (table 2).


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Discussion

We describe here the identification of a cell-surface receptor that can respond to both AMP and

adenosine. Although the responses to both ligands are pharmacologically similar, we consider the

receptor a new member of the P2Y family since the primary structure of the receptor protein more

closely resembles the P2Y nucleotide receptors than the P1 adenosine receptors. Moreover, the

sequence contains the conserved basic residue arginine at position 268 that has been reported to be

essential for binding the phosphate moiety of nucleotide ligands by P2Y receptors (27). The ability

of the receptor to bind the nucleoside adenosine, however, is unique among the P2Ys and may

require a broadening of the criteria for the P2Y classification.

Functionally, the receptor is able to respond to stimulation by inducing the mobilization of

calcium ions and causing the generation of cyclic AMP. This dual functionality is similar to that of

the adenosine receptor A2B (28,29) and the nucleotide receptor P2Y11 (19) both of which show

evidence of being dually coupled to activators of phospholipase C (calcium flux) and adenylate

cyclase (cyclic AMP production). For both A2B and P2Y11, the activation of phospholipase C and

adenylate cyclase is thought to be achieved through Gq class and Gs G proteins, respectively.

Because P2Y15-induced calcium flux and cyclic AMP generation are both insensitive to pertussis

toxin, which inactivates Go and Gi classes of G proteins, P2Y15 is likely to be similarly coupled to

the pertussis toxin-insensitive Gq class and Gs G proteins. The nature of the P2Y15’s coupling to

adenylate cyclase-stimulatory Gs proteins in our system is not completely straightforward, however,

since detectable levels of cyclic AMP production could only be seen in the presence of the adenylate

cyclase activator forskolin. This apparent requirement for forskolin, which synergistically

potentiates the cyclic AMP production, may be specific to the HEK293 host cells used, but in any

case is reminiscent of the forskolin potentiation that has similarly been reported for β-adrenergic

receptors (30).

The similarity to other receptors, particularly A2B which can respond to adenosine as a ligand


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and which is expressed on the HEK293 host cell used in the experiments presented here, was a

potentially confounding factor in our analysis and therefore had to be taken into account when

interpreting our results. In calcium mobilization experiments, despite previous reports of HEK293

calcium responses generated upon stimulation with adenosine (28,29), the response of endogenous

receptor to AMP and adenosine in our HEK293 cells was so slight as to be negligible. The

transfection procedure itself also appeared to have no influence on the calcium response since

transfectants generated with P2Y8 instead of P2Y15 also showed negligible responses to AMP and

adenosine. On the other hand, in cyclic AMP production assays, nontransfected cells gave a

response to adenosine that was indistinguishable from that seen in the transfectants. Although it was

clear that AMP could cause a distinct increase in cyclic AMP production in the P2Y15 transfectants,

we cannot say with certainty that adenosine stimulation of P2Y15 had the same effect. Confirmation

of the ability of adenosine to stimulate P2Y15 to activate adenylate cyclase must therefore await the

successful functional expression of P2Y15 in a cell type that does not express A2 adenosine

receptors.

In receptor binding assays, while AMP clearly showed saturatable binding kinetics only in P2Y15

transfectants, adenosine was able to bind specifically to receptors on both nontransfectants and

P2Y15 transfectants, albeit at a higher level in the transfectants. Competitive binding experiments

using cold AMP to compete against labeled adenosine binding, however, showed that adenosine

binds to sites on P2Y15 transfectants that can be competed with AMP, presumably P2Y15 receptors,

but on the nontransfectants binds only to sites that cannot be competed by AMP, such as other

adenosine receptors, giving compelling evidence that adenosine binds to P2Y15. Although the

affinity of the receptor for AMP and adenosine is relatively low, the ligand binding results we

obtained are in line with the range of Kd values that have been reported so far for some of the other

adenosine receptors and P2Y receptors. For example, agonist binding to the A2B adenosine receptor

is typically in the double digit micromolar range (1) and [35S]ATP[γS] binding to P2Y receptors on


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tracheal gland cells has been reported as 2.5 µM and 20 µM (31). Furthermore, the lack of

antagonism of the P2Y15 receptor by DPCPX and alloxazine, specific antagonists of A1 and A2B

receptors respectively, and the lack of significant agonism of the P2Y15 receptor by N6-

cyclopentyladenosine, CGS-21680 hydrochloride, and Chloro-IB-MECA, specific agonists of A1,

A2A, and A3 respectively, strongly suggest that the signaling responses measured were generated

through activation of P2Y15 and not another adenosine receptor.

Nevertheless, a concern when conducting experiments with AMP is the potential for enzymatic

breakdown of the molecule to adenosine. In most reports that have demonstrated the activity of

AMP, for example as an inducer of bronchoconstriction in the lungs of patients with asthma (3,32)

or as a paracrine activator of intestinal chloride ion secretion produced by neutrophils and

eosinophils (11,33), due to the absence of a receptor for AMP to which this activity could be

attributed, it has been assumed that AMP is converted to adenosine before the resultant effects are

produced. Our analysis of AMP to adenosine conversion rates did not show any evidence of

enhanced AMP breakdown in P2Y15-expressing cells. To determine, therefore, whether AMP itself

can bind P2Y15 or must first be converted to adenosine, we performed receptor binding assays

using 32P-labeled AMP and found specific, saturatable binding to the transfectants. Since the

radiolabel on the molecule would be lost upon dephosphorylation and conversion to adenosine, our

binding assay shows that AMP can bind to P2Y15 without conversion.

The expression of P2Y15 in mast cells, the respiratory tract, and kidney is consistent with effects

that have been reported for AMP, adenosine, and adenosine antagonists in these tissues. In the

respiratory tract, an immediate bronchoconstriction is typically experienced by patients with asthma

upon the inhalation of AMP or adenosine (3), and adenosine has been found to be increased in the

bronchoalveolar lavage fluid from airways of patients with chronic inflammatory conditions of the

lung, such as asthma and chronic obstructive pulmonary disease (34). Recently, adenosine, or more

commonly AMP which is more easily solubilized, has been utilized in bronchoprovocation tests for


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the diagnosis and monitoring of asthma (3,32) due to the tests’ better disease specificity and

correlation with inflammation state than other bronchoprovocators. Conversely, adenosine receptor

antagonists, such as theophylline, have been used for over 50 years as effective bronchodilators (15).

Evidence on the mechanism of adenosine and AMP mediated bronchoconstriction has indicated an

extracellular site of action and the stimulation or potentiation of mast cell mediator release (35).

Recent studies to determine which adenosine receptor is responsible for the bronchoconstriction

response, however, have failed to conclusively implicate a particular receptor, and on the contrary,

experiments in rats have ruled out the role of any of the known P1 adenosine receptors, leading to

the conclusion that an unknown receptor must be involved (36). While it is still possible that the

effects of AMP in the human respiratory tract are dependent upon its breakdown to adenosine and

the subsequent stimulation of P1 adenosine receptors, with the evidence provided here to show the

existence of an AMP receptor, the alternative possibility of a direct effect by AMP must now also be

considered. Regarding the expression in the kidneys, adenosine antagonists such as theophylline

and caffeine are well known to possess diuretic effects. These effects are thought to be mediated

primarily through the A1 and A2A adenosine receptors, but the pharmacology of these two

receptors cannot satisfactorily account for the effects seen (37). The existence of a third receptor in

the kidneys that can be affected by adenosine antagonists may help to explain the effects.

Adenosine receptor antagonists, such as theophylline, enprofylline, and caffeine, are among the

world’s most widely used drugs. Their molecular mechanism, however, remains undefined, as do

their sites of action, which include adenosine receptors, phosphodiesterases, histone deacetylases,

and other sites that have yet to be found (38,39). To determine whether P2Y15 is a target of such

antagonists, several adenosine receptor antagonists were tested and all but two showed antagonist

activity against P2Y15. The concentration of theophylline at which calcium responses could be

inhibited (Ki of 0.7 µM against AMP; 5.6 µM against adenosine) is well below that considered to be

the therapeutically optimal plasma concentration (usually 55-110 µM) (15) for this drug. Similarly,


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plasma concentrations of caffeine as low as 5 µM have been shown to relieve histamine-induced

bronchoconstriction (40), and at this concentration caffeine can effectively inhibit AMP-induced

calcium responses (Ki of 3.3 µM against AMP). Unexpectedly, however, the antagonists often

showed a large difference in the concentrations required to block AMP-mediated signaling

compared with those required to block adenosine-mediated signaling, even though AMP and

adenosine show similar EC50 values for signaling and similar Kd values for binding. The underlying

reason for this is unclear, but the discrepancy suggests that the mechanism of action of the

antagonists is more complex than can be explained by simple affinity based competition.

The identification of a receptor that binds and responds to both AMP and adenosine and is

susceptible to blocking by adenosine receptor antagonists can help us to better understand the

complex physiological effects of AMP and adenosine. Since the safety and effectiveness of

adenosine antagonists in treating respiratory diseases and other ailments has been limited by

toxicity in the central nervous system and heart (41) where most adenosine receptors are found, it

will be of considerable interest to determine whether P2Y15 can be specifically targeted in order to

develop better treatments.


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Acknowledgements

The authors of this article would like to thank Eiko Okazaki for her technical support.


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Figure legends

Figure 1. Alignment of human, mouse, and rat P2Y15 amino acid sequences. Identical residues

are shown with white backgrounds, similar residues with gray backgrounds, and dissimilar residues

with black backgrounds. Identity between either of the rodent sequences and the human sequence is

86%, and between the mouse and rat is 96%. Seven transmembrane regions as predicted by the

computer program TMpred (42) are indicated with heavy overlines and numbered TM1-7. The

Genbank accession number of the rat P2Y15 sequence is AY191367.

Figure 2. Unrooted phylogenetic analysis comparing the P2Y15 protein sequence with

adenosine receptors, other P2Y receptors, and closely related GPCRs. Phylogenetic analysis

was performed with the Neighbor Joining algorithm and the dendrogram drawn with the computer

program Vector NTI. Known ligands are indicated.

Figure 3. Tissue and cellular distribution of P2Y15. Expression in various human tissues (a) and

cells (b) was assessed by quantitative RT-PCR. The x axis represents the approximate number of

copies of messenger RNA transcript per 10 ng of total RNA after normalization to a set of five

housekeeping genes. (c) Expression of P2Y15 and the P1 adenosine receptors (gene names

ADORA1, -2A, -2B, and -3) in various leukocyte subsets was analyzed by hybridization of cRNAs

generated from cellular total RNAs to microarrays containing approximately 20,000 gene-specific

probes. The x axis represents the relative fluorescence intensity of cRNAs bound to each gene-

specific probe.

Figure 4. P2Y15 is a functional receptor for AMP and adenosine. AMP (filled symbols) and

adenosine (open symbols) stimulated calcium mobilization (a) and cyclic AMP generation (b) in a


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dose-dependent manner in transfected HEK293 cells expressing P2Y15 (circles). Nontransfected

HEK293 cells (squares), by contrast, showed little calcium mobilization in response to either ligand,

and showed cyclic AMP generation in response to adenosine but not to AMP. Calcium mobilization

was measured in 10-well replicates in an FDSS6000 functional drug screening system and plotted

as the integral of the ratio of signal to background over a 60 second time period. Cyclic AMP

generation in the presence of 10 µM forskolin was measured in duplicate with the Tropix cAMP

screen. The data for cyclic AMP generation are representative of three separate experiments. (c) No

differences could be seen between conversion rates of AMP to adenosine by nontransfected

HEK293 cells (white bars) and P2Y15-expressing cells (black bars). 1.85 µM [2-3H]-AMP was

added to triplicate cultures of nontransfectants and P2Y15 transfectants then recovered from the

medium after incubation times of 5 and 60 minutes. Thin layer chromatography analysis showed

only two bands, which migrated together with AMP and adenosine standards, indicating minimal

conversion to other products such as IMP, ADP, or ATP.

Figure 5. P2Y15 can bind both AMP and adenosine. Specific binding of increasing

concentrations of [2-3H]-adenosine (a) and [32P]-AMP (b) to transfected HEK293 cells expressing

P2Y15 (circles) and nontransfected HEK293 cells (squares) is shown. The binding of [2-3H]-

adenosine (c) or [2-3H]-AMP (d) to P2Y15-transfected cells and nontransfected cells could be

competed in both cases by unlabled AMP (filled symbols) or adenosine (open symbols). All binding

assays were performed in quadruplicate.

Figure 6. ADA can inhibit adenosine-induced but not AMP-induced P2Y15 signaling. Cyclic

AMP production induced by 25 µM adenosine in transfectants expressing P2Y15 was effectively

inhibited in a dose-dependent manner by ADA. Cyclic AMP production in response to 100 µM


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AMP, however, showed little change even at ADA concetrations up to 4 U/ml. Cyclic AMP

production was measured in 4-well replicates in the presence of 10 µM forskolin as described in

figure 4.

Figure 7. Antagonists of adenosine block AMP- and adenosine-induced P2Y15 signaling.

Calcium mobilization induced by 10 µM AMP (a) or 10 µM adenosine (b) was effectively blocked

in a dose-dependent manner by the indicated adenosine receptor antagonists. Curves for alloxazine

and DPCPX, which showed agonistic effects in both transfectants and non-transfectants, are

excluded. Calcium mobilization was measured in 6-well replicates as described in figure 4. The data

are representative of three separate experiments.


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Table 1. Ki values for antagonists of AMP- and adenosine-stimulated Ca2+ mobilization derived by

non-linear regression of the curves shown in figure 6a and 6b.

Adenosine receptor AMP antagonism Adenosine antagonism

Antagonist selectivity Ki (nM) Ki (nM)

Theophylline Non-selective 771 5,620

Caffeine Non-selective 3,309 23,100

IBMX Non-selective 540 23,800

8-SPT Non-selective 200 962

8-PT A1 384 6,330

DPCPX A1 - -

CSC A2A 277 18,100

Enprofylline A2B 3,033 64,060

Alloxazine A2B - -

Abbreviations: IBMX, 3-isobutyl-1-methylxanthine; 8-SPT, 8-(p-sulfophenyl)theophylline; 8-PT, 8-

phenyltheophylline; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; CSC, 8-(3-chlorostyryl)caffeine;

-, no detectable antagonistic effect.


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Table 2. EC50 values for P2Y15 agonist-stimulated Ca2+ mobilization.

Agonist

Adenosine receptor

selectivity EC50 (nM)

AMP 920

Adenosine 670

NECA Non-selective 6,959

N6-cyclopentyladenosine A1 72,000

CGS-21680 hydrochloride A2A -

Chloro-IB-MECA A3 -

Abbreviations: NECA, 5’-(N-ethylcaboxamido)adenosine; Chloro-IB-MECA, 2-chloro-N6-(3-

iodobenzyl)-adenosine-5’-N-methyluronamide; -, no detectable agonistic effect.


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Hisayo Inbe, Shinichi Watanabe, Miwa Miyawaki, Eri Tanabe and Jeffrey A. Encinasadenosine

Identification and characterization of a cell-surface receptor, P2Y15, for AMP and

published online March 4, 2004J. Biol. Chem.

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