Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5113-5117, June 1993 Pharmacology

Expression cloning of an ATP receptor from mouse neuroblastoma cells

KEVIN D. LUSTIG, ANDREW K. SHIAU, ANTHONY J. BRAKE, AND DAVID JULIUS* Department of Pharmacology, Programs in Cell Biology and Neuroscience, University of California, San Francisco, CA 94143 Communicated by Stephen Heinemann, March 1, 1993

ABSTRACT Extracellular ATP activates cell-surface me- A more complete characterization ofthis putative family of tabotropic and ionotropic nucleotide (P2) receptors in vascular, P2 purinergic receptors has been hampered by the complete neural, connective, and immune tissues. These P2 receptors lack of specific P2 receptor antagonists, radioligands, and mediate a wealth of physiological processes, including nitric cloned receptor cDNAs. To circumvent this difficulty, we oxide-dependent vasodilation of vascular smooth muscle and have used a Xenopus laevis oocyte expression cloning strat- fast excitatory neurotransmission in sensory afferents. Al- egy (19, 20) to isolate a cDNA encoding a functional P2U though ATP is now recognized as a signaling molecule, the receptor from NG108-15 neuroblastoma x glioma hybrid cellular and molecular mechanisms underlying its actions have cells.t We have examined the agonist selectivity, signaling been difficult to study due to the absence ofselective P2 receptor properties, species origin, and tissue distribution of the antagonists and cloned receptor genes. Nonetheless, five mam- cloned P2U receptor. malian P2 receptor subtypes have been tentatively assigned based solely on agonist specificity and signaling properties. EXPERIMENTAL PROCEDURES Here we report the cloning of a mouse cDNA encoding a P2 receptor that shares striking homology with several G protein- Expression Cloning. NG108-15 cells were grown in mono- coupled peptide receptors. When expressed in Xenopus laevis layer culture (21), total cellular RNA was isolated by the oocytes, the cloned receptor resembles a metabotropic P2U guanidine thiocyanate method (22), and poly(A)+ RNA was receptor; activation by either ATP or UTP elicits the mobili- selected on oligo(dT)-cellulose. A directional cDNA library zation of intracellular calcium. mRNA encoding the P2U puri- (2 x 106 recombinants) was constructed in pCCM6XL as nergic receptor is found in neural and nonneural tissues. described (23). pCCM6XL is a derivative of pCDM6XL in which a 778-bp Taq I fragment containing a gene encoding chloramphenicol acetyltransferase was inserted into the There is considerable evidence suggesting that ATP functions BstBI site of the supF gene. Initially, the NG108-15 cDNA as an extracellular signaling molecule in neural and nonneural library was subdivided into 10 pools of 2 x 105 clones. mammalian tissues (1, 2). In central and peripheral synapses, Templates for in vitro transcription were prepared by linear- ATP mediates fast excitatory neurotransmission (3, 4). In the izing plasmid DNA isolated from these pools with Not I. autonomic nervous system, ATP is a major purinergic Complementary RNA (cRNA) transcripts were synthesized cotransmitter that is often colocalized in secretory vesicles using SP6 RNA polymerase as described (20). with norepinephrine or acetylcholine (5, 6). In the vascular X. laevis oocytes were surgically isolated (24) and enzy- system, aggregating platelets secrete ATP and ADP, which matically defolliculated by incubation with 2 mg of collagen- stimulate the release of nitric oxide and other vasodilators ase per ml for 2 hr at room temperature. Defolliculated from the endothelium (7). In the immune system, ATP oocytes were washed five times with modified Barth's solu- modulates macrophage phagocytosis (8) and mast cell de- tion [MBS1: 7.5 mM Tris, pH 7.6/88 mM NaCl/1 mM granulation (9). In the human airway epithelium, ATP stim- KCI/2.4 mM NaHCO3/8.2 mM MgSO4/0.33 mM Ca(NO3)2/ ulates transepithelial ion transport (10), an effect that may 0.4 mM CaCl2/100 units of penicillin per ml/100 tg of underlie the therapeutic effect of ATP and UTP in the streptomycin per ml/2% Ficoll-400] and maintained in MBS1 treatment of cystic fibrosis-related lung disease (11). at 18°C. On the following day, oocytes were injected with 50 It has been postulated that these responses to extracellular nl of NG108 cRNA transcripts (=1 ug/,ul) and incubated at ATP are mediated by specific plasma membrane receptors, 18°C for 2 days prior to analysis. Voltage-clamp recording called P2 purinergic receptors (12, 13). Based on agonist was performed using a single electrode (Axoclamp 2A) in selectivity and signaling properties, five subclasses of P2 dSEVC mode. cRNA from 1 of the 10 pools rendered the receptor have been tentatively defined: three subclasses of oocytes responsive to ATP and UTP (not shown). A sib receptors (P2T, P2U, and P2y) that are believed to signal selection procedure was used to progressively subdivide this through G proteins, one subclass (P2X) that is believed to be positive pool into smaller pools of 20,000, 2000, 200, and 10 a ligand-gated cation channel, and one subclass (P2z) that is clones, finally yielding a single clone, called pP2R. Both present on mast cells, macrophages, and fibroblasts, but strands of the cDNA insert were sequenced using the dide- whose signaling mechanism is less well understood (1, 14- oxynucleotide chain-termination method (25). 16). G protein-coupled P2 receptors are found in numerous 45Ca2+ Release Assay. Defolliculated oocytes were micro- cultured cell lines, where they have been shown to activate injected with 0.5 ng ofpP2R cRNA transcripts and incubated signal transduction systems that involve the breakdown of at 18°C. After 48 hr, the oocytes were washed four times with membrane phospholipids and the elevation of cytoplasmic 5 ml of MBS2 (Ca2+-free MBS1 containing 0.1% bovine free Ca2+ (17, 18). Ionotropic P2X receptors carry Na+, K+, and Ca2+ currents and appear to be predominantly expressed Abbreviations: cRNA, complementary RNA; ATP[,yS], adenosine in neural and neuromuscular tissues (16). 5'-[y-thio]triphosphate; 2-MeSATP, 2-methylthioadenosine 5'- triphosphate; AMP-PCP, adenosine 5'-[f3,'y.methylene]triphosphate; AMP-CPP, adenosine 5'-[a,4-methylene]triphosphate. The publication costs ofthis article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L14751). 5113 Downloaded by guest on September 24, 2021 5114 Pharmacology: Lustig et al. Proc. Natl. Acad. Sci. USA 90 (1993) serum albumin) and incubated for 3 hr at room temperature RNA from NG108-15 cells, bath-applied ATP or UTP (1 mM) in 0.5 ml of MBS2 containing 30 ,uCi of 45CaC12 (1 Ci = 37 evoked inward currents (between 20 and 200 nA) that are GBq). Labeled oocytes were washed 10 times with 5 ml of typical of the activation of phospholipase C-coupled recep- MBS1 and incubated in 3 ml ofMBS1 for 90 min. Each oocyte tors (not shown). To identify a cDNA encoding the P2R, was then separately placed into 100 ,ul of MBS1 in a well of pools of 2 x 105 individual clones from a NG108-15 cDNA a 96-well polystyrene plate. After 20 min at room tempera- library were transcribed in vitro and the resultant cRNA was ture, individual oocytes were transferred to 100 ,ul of fresh injected into oocytes. The pool that rendered the oocytes MBS1 containing the indicated concentration ofnucleotide or responsive to ATP or UTP was progressively subdivided to nucleoside. The medium was sampled 20 min later and the obtain a single plasmid clone called pP2R (Fig. 1). radioactivity in the sample was quantitated by liquid scintil- Predicted Structure of P2R. Sequence analysis revealed lation counting. EC50 values (concentrations of agonists that that the cloned P2R cDNA has a 1119-bp open reading frame result in half-maximal stimulation) were determined by non- (Fig. 2), 269 bp of 5' untranslated sequence, and -1 kb of 3' linear regression analysis. untranslated sequence. The largest open reading frame en- Northern Analysis. Poly(A)+ RNA was isolated from cell codes a putative protein of 373 amino acids with a predicted lines or mouse tissues and electrophoresed in 0.8% agarose/ relative molecular mass of 42,172. Analysis of the deduced formaldehyde gels (26). The mRNA was transferred to Hy- amino acid sequence suggests that the protein contains a bond-N nylon membranes (Amersham), prehybridized for 6 small N-terminal extracellular domain, seven hydrophobic hr, and then hybridized overnight at 42°C in solution con- transmembrane domains, and a large C-terminal intracellular taining 50% formamide with 6x SSC (lx SSC = 0.15 M domain. There are two consensus N-linked glycosylation NaCl/15 mM sodium citrate). The probe was prepared by sites near the N terminus and several potential phosphory- random prime-labeling (Boehringer Mannheim) of a 2.2-kb lation sites near the C terminus (Fig. 2). The only site that Xba I DNA fragment containing the entire coding region of resembles a consensus nucleotide-binding motif [G-(X)4-G- P2R. Filters were washed in O.lx SSC containing 0.1% SDS K, ref. 27] is comprised of residues 18-24 (GDELGYK) at 65°C and exposed to Kodak XAR film with two intensifying located in the putative N-terminal extracellular domain. screens at -80°C for 2 days for cell lines and 4 days for Sequence comparisons (Fig. 3) indicate that the cloned P2R tissues. is a member of the G protein-coupled receptor superfamily. The predicted amino acid sequence of the cloned P2R con- RESULTS tains a number of highly conserved amino acids (Asn-51, Asp-79, Leu-81, Arg-131, Pro-167, and Pro-303) that are Identification of an ATP Receptor cDNA. Using a X. Iaevis oocyte expression cloning strategy (19, 20), we isolated a ATGGCAGCAGACCTGGAACCCTGGAATAGCACCATCAATGGCACCTGGGAGGGGGACGAA 329 cDNA clone encoding a P2 receptor (P2R) from NG108-15 M A A D L E P W * S T I g G T W E G D E 20 CTGGGATACAAGTGTCGCTTCAACGAGGACTTCAAGTACGTGCTGTTGCCCGTGTCCTAT 389 cells, a mouse N18TG2 neuroblastoma x rat C6 glioma cell L G Y K C R F N E D F K Y V L L P V S Y 40 hybrid cell line. In Xenopus oocytes injected with poly(A)+ GGCGTGGTGTGCGTGCTCGGGTTGTGCCTGAACGTCGTGGCTCTCTATATCTTCCTATGC 449 G V V C V L G L C L N V V A L Y I FL C 60 ADP 2-MeSATP ATP CGCCTCAAAACCTGGAACGCCTCCACCACCTACATGTTTCACCTGGCAGTTTCGGACTCT 509 R L K T W N A S T T Y M F H L A V S D S 80 CTCTACGCAGCGTCCCTGCCGCTGTTGGTTTATTACTACGCCCGGGGTGACCACTGGCCA 569 L Y A AS L P L L V YY Y A R G D H W P 100 TTTAGCACGGTGCTCTGCAAGCTGGTGCGTTTCCTCTTCTACACCAACCTCTACTGCAGC 629 F S T V L C K L V R F L F Y T N L Y C S 120 ATCCTCTTCCTCACCTGCATCAGCGTGCACCGGTGCCTGGGAGTCCTGCGCCCTCTGCAC 689 I L F L T C T S V H R C L G V L R P L H 140 AMP-PCP AMP-CPP UTP TCCCTGCGTTGGGGCCGGGCCCGTTATGCCCGCCGGGTGGCTGCGGTTGTGTGGGTGCTG 749 S L R W G R A R Y A R R V A AVV W V L 160 GTGCTGGCCTGCCAGGCACCCGTGCTCTACTTCGTCACCACCAGCGTGCGGGGAACCCGG 809 V L A C O A P V L Y F V T T S V R G T R 180 ATCACTTGCCATGACACCTCGGCCCGAGAGCTCTTTAGCCATTTTGTGGCTTACAGCTCC 869 I T C H DT S A R EL F S H F VA Y S S 200 GTCATGCTGGGTCTGCTTTTTGCTGTGCCCTTTTCCGTAATCCTGGTCTGTTACGTGCTT 929 V M L G L L F A V P F S V I L V C Y V L 220 ADO ATPyS ATGGCCAGGCGGCTGCTCAAACCGGCTTATGGGACCACAGGAGGTCTGCCTCGGGCCAAG 989 M A R R L L K P A Y G T T G G L P R A K 240 CGCAAGTCTGTGCGCACCATTGCCTTGGTACTGGCCGTCTTCGCCCTCTGCTTTCTGCCT 1049 R K S V R T I A L V L A V F A L C FL P 260 TTCCACGTCACGCGCACCCTCTACTACTCCTTCCGATCACTTGACCTCAGCTGCCACACC 1109 F H V T R T L Y Y S F R S L DL S C H T 280 CTCAACGCCATCAACATGGCATATAAGATCACCCGGCCGCTGGCCAGCGCCAACAGTTGT 1169 L N A I N M A Y K I T R P L A S A N S C 300 Vf < 200 nA CTTGACCCGGTACTCTACTTCCTGGCAGGGCAGAGACTTGTCCGCTTTGCCCGAGATGCC 1229 L D P V L Y FL A G Q R L V R F A R D A 320 lOs AAGCCACCCACGGAGCCTACCCCCAGCCCACAGGCTCGTCGCAAGCTGGGCCTGCACAGG 1289 K PP T E P T P S P Q A R R K L G L H R 340 FIG. 1. Nucleotides elicit inward currents in Xenopus oocytes CCTAACAGAACTGTGAGGAAAGATTTGTCAGTCAGCAGTGACGACTCAAGACGGACAGAG 1349 injected with 0.5 ng of cRNA transcribed in vitro from pP2R. The P NR T V R K D L S V S S D D S R R T E 360 oocytes were single-electrode voltage clamped 48 hr after cRNA TCCACACCAGCTGGAAGTGAGACTAAGGACATTCGGCTATAG 1391 injection and held at -60 mV while superfused with MBS1. Three S T P A G S E T K D I R L - 373 original current traces are shown, and the thick lines above the traces indicate the length of time the oocytes were incubated with the FIG. 2. The nucleotide sequence of the largest open reading following compounds (50 uM): ATP, UTP, adenosine 5'-[y- frame of the cloned P2R is shown above the deduced amino acid thio]triphosphate (ATP[yS]), 2-methylthioadenosine 5'-triphosphate sequence. A putative nucleotide-binding region in the N-terminal (2-MeSATP), adenosine 5'-[,8,-methylene]triphosphate (AMP- domain is indicated by a black bar, two potential N-linked glycosy- PCP), ADP, adenosine 5'-[a,4-methylene]triphosphate (AMP-CPP), lation sites are indicated by asterisks, and the seven putative and adenosine (ADO). transmembrane domains are underlined. Downloaded by guest on September 24, 2021 Pharmacology: Lustig et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5115

P2 M Ar D. E W ------r S 1 0 THROMBIN MGPRRLLLVAACFSLCGPLLSARTRARRPESKATN N T D RS F LL RN PUND 50

P2 TINGT|i GD LGYKC ------E 31 THROMBIN KY E P F DEW KNESGLTEYRLVSINKSSPLQKOLPAFISEDA S-0FRGY L TS SW 1 00 PAF MELNSSS ------EV D S EH 1 3 ANGII M I L N SST - -- EDGIKRIQDDCPKAGRHNY 26 GPRN1 MDLHLFDYAEPGNFSD SWPCNSSD-----C VVDTVMCPNMPNKSV 42 IL-8 MEVNVWNMTOLWTWFEDEFANATGMPPVEKDYSPCLVVTQTLjNjKYV 46

P2 K tL V S GVfO Vt V lIP--ORLf8VAASTT TiFHLAVS 79 THROMBIN L T L FV SV 1TVTF VS P I4I VVj L K V K K PA VVA} -LLATAD 1 48 PAF RA0T8 kIIF V S F A NGYWVW: AR YPt K LE E K IF V A 63 ANGII F M T L S F VV IF NS L V VI V I- YFY K L KTVASV F L LN :L ALA D 74 GPRNI L L YTdS'FII:Y I F I FLILM4] L[VMVWV -- IQAK GY D THOC'- .LN,jJAI A D 90 IL-8 VQV SA LL A L L S L S L V M L V -L NYSR MNLRSDVTDVLL LN -A MA 0 90 II III P2 SRY9ASIFfLEI YYARDHWPFS'TVLK 17T1~~~ 1 2 9 THROMBIN V F V S V PK SY FSSD GS E.A T AF Y LL 198 PAF L F L TILPTJW VY-SNGGNN FLLP K CAOFIG S ITY 11 3 M 124 GPRN1ANGII IL-B LWVVLVLOFL..JL...IL LLTWP2JWT N4dIWAVS-PIW9VSLVQHNWAV\ TAMEYAWP~~~~~~~~K E WMGK~~~MGEL~N4 TCKVT-A SHAFLFTNLV0$...... L.....VALSVYIVKEVsSFINLAiTISIFALTIVNFSLL:LAO 140 IL-8 L[gF[RL T 1WAVS KE K G ILSffP> }<|ES L VK E V S L A 1 38 IV P2 H CL GL PLHL G(fa A R 9RA A.LVL. Q AP-VYF VT TSR 1 77 THROMBIN DFY:A:: V P|MQ$WS-R T L G ASF T C ALA IGAVV L E VPP 247 PAF N|F OA LK Y|PIIKTAQATT R K R G A L S L0V I: V EAA S Y F L S TNV VS N 1 63 ANGII D YLAIVHMK[gR MLTML VKV T C II L- A GL A S LFT I H RN V F F E 1 73 GPRN1 LYIWS T YFTNT P S S RKK MV VE|C L FJl-HL - A V S LlPID T YL KQ|V TS A 1 89 IL8 D:YLSITYFTNTPSSTKKMHVAVFICLGI LAFSLVSLPIDTS0RVFS 1 85 v P2 GTRI-TC.H:D...... B|TSAAER 225 A AL F-dHFOAYSSVMLGWL(TT...... *.-V---P-T-SVT3-VL THROMBIN L NTT CHDV L N E TLEGYYA F SA V F L S T CYVS CL 297 PAF K A G S G NI T R C F E H Y E K G00K PL II H C VIF V FL LI LLFC LV H TIL 21 3 ANGII NONET VA F H Y E S NS T -L PUG L G L T KN I4FLf JLIITSfT W K TL 222 GPRN1 S NN E T R S FYPEHS KEWL GMEL92SVVIFAVQ IHELJYIF LLAI 239 IL-8 NNSSPVCYEDLGHNTAK-WRMVLRI LPHT F GTGFTLbTg 234 VI P2 E3PlGTT GG LP R A K S V[qTB | .FAL FFX t T R Tld - - Y ::F R S 273 THROMBIN S S SWA N --- - R S KSR A L F LSWAIVF. IlI L L ITA-L - HL:!j- 339 PAF M0 P VK a - Q RN A E V R R A L W M V C T|V L A V FlV OFV HjPV QL P W T L A E L G M 262 - F K AV L F SW FTFMDVL L G L 270 ANGII K O KNK P UD D ITL FF VPH Q I IQ E.E - -- GPRN1 S A - S SD 0O- E K HSSR- - K IF S Y V V F L V: A V LQ|D F S L H Y 282 IL-8 FQ -HMG - QKHRAM- [-VI.!|FA4V L I LYYN LV L L A DT L MRT HV 277 VII P2 322 THROMBIN W.SHTSTLI -EAAVFAY.LLCVCYVSSS 1...... -.Y ASSECYVYSIL 387 PAF 310 WP--SSN HLAD K L L$TN D IF LTKKFRH LSEK L N I ANG II 31 9 FTARL E H A LF TA L H V MPTICX^TOOLC S L V HO V NBIP---t-I IN RN FF 332 GPRN1 IP I~~~~~~~~~~~~IL-kN F N YYSF~| F L G K K YIRF KLKIYflLY V 0 L L5Y IL-8 GET GANDR1§IC .IDAA8 L DA -T- E I L GFLHSOLNI1FVF1!A TtI GQN F R NKGHL K ML AA 327 FIG. 3. Sequence comparisons of P2R and G protein-coupled receptors. The deduced amino acid sequence of P2R (P2) was aligned to that of the human thrombin receptor precursor (425 amino acids, ref. 28), guinea pig platelet-activating factor receptor (PAF; 342 amino acids, ref. 29), bovine angiotensin II type I receptor (ANG II; 359 amino acids, ref. 30), GPRN1, a putative human vasoactive intestinal peptide receptor (362 amino acids, ref. 31), and rabbit interleukin 8 receptor (IL-8; 355 amino acids, ref. 32). The boxed and shaded amino acids indicate identical residues. Roman numerals and brackets denote the seven putative transmembrane domains. believed to be important for the function ofG protein-coupled and the absence of selective high-affinity radioligands. We receptors (33). The cloned receptor is most similar to recep- therefore examined the pharmacology of the cloned receptor tors for thrombin (25% identity), platelet-activating factor in Xenopus oocytes injected with pP2R cRNA transcripts. In (25% identity), angiotensin II (22% identity), interleukin 8 voltage-clamped oocytes expressing the cloned P2R, inward (23% identity), and GPRN1, a putative vasoactive intestinal currents (,s500 nA) were elicited by bath application of50 AuM peptide receptor (21% identity). The cloned receptor is ATP, UTP, or ATP[yS] but not 50 AM 2-MeSATP, AMP- substantially less similar (<12% identity) to G protein- PCP, ADP, AMP-CPP, or adenosine (Fig. 1). Agonist stim- coupled receptors for adenosine and cAMP. ulation of phospholipase C-coupled receptors in oocytes Functional Characterization of the Cloned P2R. Functional activates endogenous calcium-dependent chloride channels characterization of the cloned receptor using transfected that carry inward currents (34). Thus, ATP also elicited an mammalian cells was hindered by the presence of endoge- increase in the rate of 45Ca2+ efflux (Fig. 4), indicative of an nous P2 receptors and ubiquitous nucleotide-binding proteins increase in intracellular free Ca2+ (35). The order of agonist Downloaded by guest on September 24, 2021 5116 Pharmacology: Lustig et al. Proc. Natl. Acad. Sci. USA 90 (1993) identical to that of the pP2R PCR product, whereas the restriction map of the rat PCR product differed at one Ava I I In on Northern U)a) 11 and one Sma site (not shown). addition, C: blots, an -2.4-kb RNA species was detected in NG108-15 0 cells and mouse N18TG2 cells, whereas an -3.0-kb RNA a)cn species was detected in rat C6 glioma cells (Fig. 5). Taken 01) together, these observations demonstrate that the cloned P2R .,, cDNA is of murine origin. Northern blot analysis revealed that mRNA encoding the x P2R is widely distributed in mouse tissues. An %2.4-kb RNA species was detected in spleen, testes, kidney, liver, lung, 0\0 heart, and brain (Fig. 5). An -2.4-kb RNA species was also detected in mouse NlE-115 neuroblastoma cells.

-8 -7 -6 -5 -4 -3 DISCUSSION [Nucleotide] (log M) We have isolated a cDNA clone encoding the first member of FIG. 4. Characterization of the agonist specificity of the cloned what, to our knowledge, is likely to be a new gene family of P2R using a 45Ca2+ release assay. Xenopus oocytes expressing the nucleotide receptors. Our results now provide molecular P2R were labeled with 45Ca2+ and then incubated for 20 min with the genetic evidence for the existence of plasma membrane indicated concentration of ATP (o), UTP (o), ATP[yS] (m), 2-Me- receptors for ATP. The cloned P2R has predicted structural SATP (n), AMP-PCP (A), ADP (O), AMP-CPP (v), or adenosine (*). features that are characteristic of most known G protein- The % maximal response was determined for each experiment by coupled receptors: an N-terminal extracellular domain with dividing the cpm in each sample by the cpm in samples taken from two consensus sites for N-linked glycosylation, seven hy- oocytes treated with a maximally effective concentration of ATP. drophobic putative transmembrane a-helices, and a large The data are the mean ±SEM of three separate experiments. C-terminal intracellular domain containing several consensus potency for 45Ca2+ efflux was ATP (EC5o = 0.7 ,uM) UTP phosphorylation sites. A surprising finding is that the cloned (EC50 = 1.1 ,JM) > ATP[yS] (EC50 = 7.9 ,uM) >> 2-MeSATP P2R is considerably more homologous to G protein-coupled AMP-PCP ADP AMP-CPP > adenosine (Fig. 4). In peptide receptors than to receptors for the structurally re- uninjected or water-injected oocytes, none ofthe compounds lated ligands adenosine and cAMP. evoked inward currents or enhanced the rate of Five mammalian P2 receptor subtypes have been tenta- reproducibly tively assigned based on agonist selectivity and signaling 45Ca2+ efflux (not shown). In these control oocytes, ATP and properties (2, 15). The characteristics of the cloned P2R are UTP occasionally induced inward currents (typically <20 most similar to those of the P2u receptor subtype (15). The nA), which may be due to the activation of a P2 receptor cloned P2R and the endogenous P2U receptor expressed in endogenous to the oocytes. Of the subtypes of P2 receptors NG108-15 cells (37) are activated by ATP or UTP and both that have been tentatively assigned, this agonist specificity is couple to a signal transduction system involving increases in most similar to that ofthe P2U (nucleotide) receptor subtype, cytoplasmic free Ca2+. Our findings demonstrate that the for which ATP and UTP are more potent agonists than pharmacological profile of the P2U site can be attributed to a 2-MeSATP (15). In NG108-15 cells, ATP and UTP activate an single gene product. This direct relationship has proven endogenous P2u receptor that mobilizes intracellular Ca2+ difficult to establish due to the lack of subtype-selective (36, 37). pharmacological or molecular probes. ATP hydrolysis does not appear to be necessary for P2 receptors have been implicated in the regulation of receptor activation since the cloned P2R (Fig. 1) and the neurotransmission, vascular tone, wound healing, inflamma- endogenous P2 receptor in NG108-15 cells (37) are activated tion, muscle contraction, immune responses, pulmonary by ATP[yS], a slowly-hydrolyzed ATP analog. A search for function, and cell growth (1, 2, 7, 16). Ca2+-linked P2 recep- ATP-binding motifs in the P2R revealed one site in the tors that respond to ATP or UTP are expressed in cell lines putative N-terminal extracellular domain that resembles a consensus ATP-binding site. To determine whether this site 1 2 3 4 5 6 7 8 9 10 1I is essential for receptor activation by ATP, we constructed a k b mutant receptor in which residues 18-24 (GDELGYK) have 7.5- k b been deleted. Voltage-clamp analysis showed that oocytes -7.5 expressing this mutant receptor exhibited similar responses 4.4- to ATP and UTP as did oocytes expressing the wild-type -4.4 receptor (not shown). 2.4 Jl* :. Species Type and Tissue Distribution of the Cloned P2R. -2.4 Polymerase chain reaction (PCR) and Northern blot analyses -1.4 revealed that the receptor is derived from the mouse N18TG2 1.4- neuroblastoma and not the rat C6 glioma parent cell line. Two oligonucleotide primers corresponding to positions 277-306 and 646-675 of the P2R cDNA (Fig. 2) were used to amplify total mouse or rat genomic DNA by standard PCR. A PCR FIG. 5. Determination ofthe tissue distribution ofP2R mRNA by product corresponding to an =0.4-kb region of the P2 recep- Northern analysis. Shown is an autoradiograph ofa blot probed with tor could be amplified from pP2R, rat genomic or mouse random primer labeled DNA synthesized from an Xba I fragment of genomic DNA templates. Amplification at higher stringency, P2R. Northern blots were prepared using poly(A)+ mRNA isolated however, yielded an =0.4-kb PCR product only with pP2R from the following cells and mouse tissues: mouse NlE-115 neuro- and blastoma cells (5 ,g, lane 1), mouse N18TG2 neuronal cells (5 Ag, mouse genomic DNA (not shown). When PCR products lane 2), rat C6 glioma cells (5 ,ug, lane 3), mouse neuroblastoma x rat of the lower stringency reactions were digested with Ava I, glioma NG108-15 cells (5 ,g, lane 4), spleen (0.7 ,ug, lane 5), testes BstEII, Eae I, Pst I, or Sma I and resolved on a 1.5% agarose (2 .g, lane 6), kidney (1 ,ug, lane 7), liver (2 j.g, lane 8), lung (2 ,g, gel, the restriction pattern of the mouse PCR product was lane 9), heart (2 ,ug, lane 10), and brain (10 ,g, lane 11). Downloaded by guest on September 24, 2021 Pharmacology: Lustig et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5117 derived from many different sources, including neural, mus- 13. Burnstock, G. & Kennedy, C. (1985) Gen. Pharmacol. 167, cular, vascular, connective, and epithelial tissues. Consistent 433-440. with the idea that we have cloned a ubiquitous P2 receptor, 14. Gordon, J. L. (1990) Ann. N. Y. Acad. Sci. 603, 46-52. P2R mRNA is expressed in all mouse tissues that we have 15. O'Connor, S. E., Dainty, I. A. & Leff, P. (1991) Trends Phar- analyzed. Presumably, P2R-mediated changes in intracellu- macol. Sci. 12, 137-141. lar free Ca2+ or other cytoplasmic second messenger systems 16. Bean, B. (1992) Trends Pharmacol. Sci. 13, 87-90. promote tissue- or cell-type-specific physiological responses. 17. Dubyak, G. R. (1991) Am. J. Respir. Cell Mol. Biol. 4,295-300. ATP has been demonstrated to act as a rapid excitatory 18. Irving, H. & Exton, J. H. (1987) J. Biol. Chem. 262, 3440-3443. neurotransmitter in the nervous system and as a hormone in 19. Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M. many & Nakanishi, S. (1987) Nature (London) 329, 836-838. nonneural tissues (1, 2). Cloning of this P2 receptor 20. Julius, D., MacDermott, A. D., Axel, R. & Jesseli, T. M. (1988) provides a crucial reagent for examining the structure, func- Science 241, 558-564. tion, and expression of members of the ATP receptor family. 21. Klee, W. A. & Nirenberg, M. (1974) Proc. Natl. Acad. Sci. USA 71, 3474-3477. We thank Nila Patil, Andrew Peterson, and Svetlana Shtrom for 22. Cathala, G., Savouret, J. F., Mendez, B., West, B. L., Karin, advice about cDNA library construction and RNA isolation, Laurie M., Martial, J. A. & Baxter, J. D. (1983) DNA 2, 329-335. Erb and Gary Weisman for discussion about P2 receptors, and Henry 23. Maricq, A. V., Peterson, A. S., Brake, A. J., Myers, R. M. & Bourne for comments on the manuscript. This work was supported Julius, D. (1991) Science 254, 432-437. by the National Institutes of Health, by a National Science Foun- 24. Marcus-Sekura, C. J. & Hitchcock, M. J. M. 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