Expression Cloning of an ATP Receptor from Mouse Neuroblastoma Cells

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.

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