A cDNA clone encoding the human prostaglandin (PG). E, receptor EP, subtype has been isolated from a human lung cDNA library. The 1.9-kilobase pair cDNA, ...
Vol. 269, No. 16, Issue of April 22, pp. 11673-11877, 1994 Printed in U S A .
THE JOURNAL OF BIOLOGICAL. CHEMISTRY Q 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning, Functional Expression, and Characterization of the Human Prostaglandin E, Receptor EP2 Subtype* (Received for publication, October 11, 1993,and in revised form, January 25, 1994)
Lison Bastien, Nicole Sawyer$, Ryszard GrygorczykS, KathleenM. MettersS, and Mohammed Adam0 From the Departments of Molecular Biology and $Pharmacology, Merck Frosst Centre for Therapeutic Research, E? 0. Box 1005, Pointe Claire-Dorval, Quebec H9R 4P8, Canada mechanisms. Thus activationof the EP, receptor has been asA cDNA clone encoding the human prostaglandin (PG) E, receptor EP, subtype has been isolated from a human sociated with a rise in inositol triphosphate and intracellular lung cDNA library. The 1.9-kilobase pair cDNA, hEP,, calcium, activation of the EP, receptor with an increase in encodes for a 488-amino acid protein with a predicted intracellular CAMP, and activation of the EP, receptor with a molecular mass of 53,115and has the seven putative decrease in intracellularCAMP. The cloning of the mouse and transmembranedomainscharacteristic of G protein- human EP, subtypes(4,5), mouse EP, subtype (6),and mouse, coupled receptors.The specific bindingof [‘HIPGE, to rat, and bovine EP, subtypes (7-9) has now been reported. COS cell membranes transfected with the hEP, cDNA These recent studies have alsoshown that there aredifferent was of high affinity with an equilibrium dissociation isoforms of the EP, subtype which arise at the COOH terminus constant (K,)of 1 PM and the rank orderof potency for of the proteinas a result of alternative splicing and, at least in prostaglandinsincompetitionfor [‘HIPGE, specific the case of the bovine forms, can couple to different signal binding was PGE, = PGE, >> iloprost > PGF,, > PGD,. In competition studies using more selective prostanoid-re- transductionpathways, includingelevation of intracellular ceptor agonist and antagonists, the [‘HIPGE, specific CAMP(9).Similar isoforms of the EP, and EP, subtypes have binding was competed by MFJ28767, an EP, agonist, but not been identified. The EP, receptor is reputedly present in the vasculature, the not by theEP,-preferringantagonists AH6809 and gastrointestinal, tracheobronchial, and reproductive tracts, the SC19220, or by the EP, agonist butaprost. Electrophysikidney and also in the sphincter, dilator, and ciliary muscles in ologicalstudies of Xenopus oocytesco-iqjectedwith the eye (2, 3). Commensurate with this distribution, the EP, hEP,and cystic fibrosis transmembrane conductance regulator (CAMP-activatedC1- channel)cDNAs detected subtype hasbeen conventionallylinked withPGE,-mediated vaPGE,-specific inward C1- currents, demonstrating that sodilation and smooth muscle relaxation, maintaininghomeothe hEP,cDNAencoded a functional receptor which stasis in these tissues. Other putative physiological roles forthe produced an increase in C A M P levels. Thus, we have EP, subtype include the stimulationof secretion in the stomach cloned the human EP, receptor subtype whichis func- and small intestine, the control of intraocular pressure, and the tionallycoupledtoincreasein C A M P . Northernblot regulation of bone reabsorption. PGE, is also known to play a analysis showed that hEP, is expressed as a 3.8-kilobasecentral role in thepathophysiology of inflammation insynergy mFtNA in a number of human tissues with the highest with other proinflammatory mediators. Although the involveexpression levels present in the small intestine. ment of all t h e E receptor P subtypes needs to be delineated, the EP, subtype is thought to be in partresponsible for vasodilation, oedema formation, hyperanalgesia,modulation of the immune Prostaglandin (PG)’ E, is involved in a number of physiologi- system, and the breakdown of bone and cartilage associated cal and pathophysiological events in many tissues throughout with disorders such as rheumatoid arthritis. the body (1).The physiological actions of PGE, are mediated We report here the cloning of the human EP, receptor subthrough its interaction withcell surface prostaglandinE recep- type. The completenucleotide and deduced amino acid setor(s). There are three pharmacologically defined subtypes of quences of the human EP, receptor, as well as its functional the EP2receptor, EP,, EP, and EP, (2,3).These three subtypes expression in Xenopus oocytes and ligand binding characterisall display high affinity for PGE, but show differences in their tics, are described. The acquisition of the humanreceptor cDNA afinities for various synthetic agonists and antagonists and will allow the elucidation of the role of PGE, and the EP, signal transduction subtype in human physiological and pathophysiological states exerttheiractionsthroughdifferent and paves the way for the development of therapeutic drugsof * The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore be hereby marked use in inflammatorydiseases. “advertisement” in accordance with 18 U.S.C.Section 1734 solely to indicate this fact. MATERIALS AND METHODS The nucleotide sequence(s) reported in this paper has been submitted Human EP, Receptor cDNA Zsolation-A mouse EP, receptor partial to the GenBankmIEMBLData Bank with accession number(s) L28175. 8 To whom correspondence should be addressed: Dept. of Molecular cDNA (680 base pairs) was obtained by reversetranscriptionP-815cell total Biology, Merck Frosst Centre for Therapeutic Research, Mer& Frosst polymerase chain reaction from mouse mastocytoma Canada Inc., P. 0.Box 1005, Pointe Claire-Dorval, Quebec H9R 4P8, RNA and cloned in pBluescript SK (Stratagene).This 680-base pair fragment covered the transmembrane segment VI1 and the complete Canada. Tel.: 514-428-8546;Fax: 514-428-8615. The abbreviations used are: PG, prostaglandin; MES, &norpho- carboxyl-terminal portion of the EP, receptor protein (6). The mouse lineethanesulfonic acid; CFTR, cystic fibrosis C1- channel; kb, kilobase EP, receptor reverse transcription-polymerasechain reaction fragment was used to generate a 32P-labeledcDNA probe to screena human lung paids); IBMX, isobutylmethylxanthine. Alto, CA) using standard techniques (10). * Prostanoid receptors are designated following the recommendation A gtl0 library (Clontech, Palo of the IUPHAR Commission on Receptor Nomenclature and Classifica- A total of 4 x lo5 plaques were screened and 12 clones that hybridized tion (29). with the probe were isolated, plaque-purified, and the DNA was pre-
11873
11874
Cloning of the Human Prostaglandin E (EPdReceptor GGCACAGCCTCACACCTGAACGCTGTCCTCCCGCAGACGAGACCGGCGGGCACTGCAAAGCTGGGACTCGTCTTTGARGGAAAAAAAATA 90 GCGAGTAAGAAATCCAGCACCATTCTTCACTGACCCATCCCGCTGCACCTCTTGTTTCCCAAGTTTTTGAAAGCTGGCAACTCTGACCTC 180 GGTGTCC~TCGACAGCCACTGAGACCGGCTTTGAGARGCCGAAGATTTGGCAGTTTCCAGACTGAGCAGGACAAGGTGAAAGCAGG 2 7 0 TTGGAGGCGGGTCCAGGACATCTGAGGGCTGACCCTGGGGGCTCGTGAGGCTGCCACCGCTGCTGCCGCTACAGACCCAGCCTTGCACTC 360
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FIG.1. Nucleotide and deduced amino acid sequences of the human EP, receptor cDNA.The deducedamino acid sequence is shown below the nucleotidesequence using the single letter code. The putative transmembrane domains (I-VII) are underlined. Asterisks denote potential N-glycosylationsites and filled diamonds denote potential phosphorylation sites. An in-frame TGA in the 5'-untranslated region is double underlined.
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pared by the plate lysate method (10). One clone containing a 1.957- changes in intracellular cAMP concentration. For this assay, an oocyte kilobase pair insert was subcloned into pBluescript SK(+), sequenced co-expressingCFTR and hEP, was placed in a 0.5-ml perfusion chamber entirely on both strands using the Pharmacia T7 DNA sequencing kit, and voltage-clampedat -60 mV (with two microelectrodesof 0.5-2.0 MR and designated human EP, (hEP,). resistance filled with 3 M KCl) using a Turbo TEC 01C amplifier (NPI 'IFansient Expressionin COS-M6 Cells and Ligand Binding AssaysInstruments, Germany). Following perfusion with the ND96 solution The 1,507-kilobase pair FspI-ScaI human EP, cDNA fragment, corre- containing the required ligand the CFTR-mediated C1- current response sponding to the full-length EP, coding sequences, was subcloned into was recorded. Northern Blots-Total RNAs from various human tissues were either the EcoRV site of an eukaryotic expression vector, pcDNAl(amp) (Invitrogen) and thecorrect orientation was verifiedby PstI digestion. The obtained from Clontech orprepared by the guanidinium isothiocyanate plasmid DNA, pcDNAl-hEP,, was prepared using the Qiagen plasmid procedure (16). Total RNA (10 pg) fromvarious tissues were separated preparation kit (QIAGEN).The pcDNAI-hEP, plasmid wasthen trans- by electrophoresis on a 0.8% agarose gel containing formaldehyde and fected into COS-M6 cells using the DEAE-dextran methodwith chloro- transferred to Hybond N membrane (Amersham).The membranes were quine (11).The cells weremaintained in culture for 72h, thenharvested UV cross-linked and hybridized at 42 "C with a radiolabeled hEP, cDNA and membranes prepared by differential centrifugation (1000 x g for 10 for 20 h as described. (17). min, then 100,000 x g for 30 min) following lysis of the cells by nitrogen cavitation (12). C3H]PGE, binding assays wereperformed in 10 rn RESULTS AND DISCUSSION MES/KOH (pH 6.0), containing 1 m~ EDTA, 10 m~ MnCl,, 0.4 XIM Cloning of the Human EP, Receptor-In order to isolate the [3HlPGE,(154 Cilmmol; DuPont-NewEngland Nuclear), and 10-15 pg of protein from the 100,000 x g membrane fraction. Incubations were human PGE, receptor EP, subtype, a human lung cDNA liconducted for 45 min at 30 "C prior to separation of the bound and free brary was screened using a 0.68-kb fragment encoding the radioligand by rapid filtration through GF/B filters presoaked in 10 rn COOH-terminal 165 amino acids of the mouse EP, receptor MESKOH (pH 6.0) containing 0.01% (w/v) bovineserum albumin. FilcDNA as a probe. A number of positive clones were identified. ters were washed with 16 ml of soaking buffer and the residual [3H]PGE,bound to the filter was determined by liquid scintillation One positive phage clone containing an insertsize of 1.9 kb was counting. Specific binding was defined as the difference between total isolated, cloned, and sequenced and designated UP,. The binding and nonspecific binding which was determined in the presence cDNA sequence and the deduced amino acid sequence of the of 1 p~ PGE,. hEP, are shown in Fig. 1. The nucleotide sequence of hEP, Functional Expression of the Human EP, Receptor in Xenopus predicts an open reading frame of 1,464 base pairs which enOocytes-Oocytes were taken from adult females ofXenopuslaeuis using codes a protein of 488 amino acids witha calculated molecular standard surgical procedures (13). To remove follicle cells, oocytes were treated for 60-120 min with freshly made collagenase (2 mglml, type 2, mass of 53,115. The first ATG at position 388 has a reasonably WorthingtonBiochemicalCorp.) in Ca2+-freeND96 solution (96 rn good consensus sequence for translation initiation( A T C U T ) NaCl, 2 rn KCl, 1 m~ MgCl,, 5 m~ HEPES, 2.5 m~ sodium pyruvate, (18). The nucleotide sequence contains a 388-bp 5"untrans0.5 m~ theophylline, 50 mg/l gentamicin, d . 8 rn CaCl,, pH 7.6). De- lated leader including an in-frame TGA stop codon 69 bp upfolliculated stage 5-6 oocytes were selected and maintained in ND96 stream of the predicted start codon. solution. Oocyte nuclei were co-injected with 2.6 ngof pcDNA-CFTRand Hydropathy profile analysis (19)of the deduced protein se1.4 ng of pcDNAl-hEP, and incubated at 18 "C for 48-72h before testing. quence revealed the presence of seven hydrophobic putative The activity of the cystic fibrosis C1F channel (CFTR) is known to be regulated by cAMP-dependent protein kinase A (14). Therefore CFTR transmembrane segments, a characteristic feature of the G expression in oocytes has been used as a sensor of receptor-mediated protein-coupled receptor (20). A comparison of the amino acid
11875
Cloning of the Human Prostaglandin E (EPJReceptor
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FIG.2. Comparison of the amino acid sequences from the human and mouse EP, receptor proteins. Identities are indicated by solid lines and differences between species are markedwith the respective one letter code. Dots indicate amino acid deletions. The putative transmembrane domains (I-VII) are indicated above the sequence by a solid line.
sequences of hEP, with mouse EP, is shown in Fig. 2. The overall amino acid sequenceidentity between the two receptors is 88%.The major difference between the two receptors is the presence of an extra25 amino acids at theNH, terminus of the mouse EP, protein and a difference of six consecutive amino acids in the thirdextracellular loop. Whether these differences will result in significant functional differences betweenthe two receptors is under investigation. Despite these differences, the pharmacological properties of the hEP, receptor examined so far were similar to those of the mouse EP, receptor (see below and Ref. 6). Several characteristic amino acids, commonly observed in G protein-coupled receptors, were conserved in the human lung EP, receptor. These include the two potential N-linked glycosylation sites (atAsn7andinthe extracellular surface of the protein, 2 cysteines in the first and second extracellular loops, which may make a disulfide bond, and 4 prolines in the fourth, sixth and seventh transmembrane segments, which may contribute to the ligand-binding pocket (21). Furthermore, a conserved arginine (at position 316) present in the seventh transmembrane segment of all known prostanoid receptors is also found in the hEP, receptor. This basic residue may be involved in the binding of prostaglandins which are acidic in nature (22). Finally, there are multiple serine and threonine residues which may serve as potential sites of protein kinase phosphorylation located throughout the COOH terminus and third cytoplasmic loops. Phosphorylation events are proposed to be involved in receptor desensitization (23). Human EP, cDNA Expression in COS-M6 Cells-In order to validate that thecDNA described in thisreport did indeed code for the hEP, receptor, [,H]PGE, binding assays were performed using membranes prepared fromCOS-M6 cells transfected with pcDNAl-hEP, to determine the affinities of prostaglandins and related synthetic analogs for this receptor subtype. The most effective competing ligands were PGE, and PGE, which wereequipotent with IC,, values of 1m. The rank order of affinity for prostaglandins, including the metabolically stable prostacyclin analog iloprost, was as predicted forthe EP receptor from previous pharmacological studies, with PGE, = PGE, >> iloprost > PGF,, > PGD, (2) (Fig. 3A). Several EPreceptor subtype selective ligands were also evaluated in competition binding studies (Fig. 3B). The EP, subtype-selective antagonists AH6809 and SC19220, whichdisplayed IC,, values of approximately 0.5 and 6.7 p,respectively, at the hEP, receptor (51, were both inactive at thehEP, receptor. In addition,
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[Competingligand] @M) FIG.3. Competition for [SHIPCE,specific binding to pcDNAlhEP, transfected COSMB membranes. f3H1PGE,binding assays were performed as described under "Materials and Methods." Panel A, the percentage maximum f3H1PGE,specific bindingat various concentrations of competing prostaglandins and prostaglandin analogs was iloprost (m), ( O ) , y d PGD, ( ). determined. PGE, (O),PGE, (O), Panel E , the percentage maximum C3H1PGE, specific bmdlngat vanous concentrations of EP-selective receptor agonist or antagonists. MB28767 (O),butaprost (O), AH6809 (m), and SC19220 (0).
PGF.,
the EP, subtype-selective agonist MB28767 had an IC,, value of approximately 9 rm and was therefore 30-fold less potent at the hEP, receptor than the hEP3a receptor (15). These differences in selectivity substantiate that we have cloned the hEP, receptor. It is of interest, however, that butaprost, which has been definedas anEP,-selective agonist (24), hadan IC,, of 30 p at the hEP, receptor and was, therefore, essentially inactive. This is in agreement with data obtained from similar competition studies with the mouse EP, receptor where butaprost was inactive at 10 p (6). Functional Expression of the Human EP, Receptor in Xenopus Oocytes-Functional activity was determined in oocytes co-expressing CFTR and hEP, using voltage-clamp techniques, as described under "Materials and Methods." Each oocyte was first challenged with 3 nw 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, which resulted in an elevation of CAMP due to basal adenylate cyclase activity (25). IBMX-induced CAMPelevation in turn activated CFTR resulting in aninward C1- current (26), which is shown in Fig. 44 as a downward deflection. The large IBMX-induced current (700 nA) confirmed that CFTR was expressed in each individual oocyte.IBMX was then removed by washing and the oocyte
Cloning of the Human Prostaglandin E (EPJReceptor
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FIG.4. Expression of a functional human EP, receptor in Xenopus oocytes. A, an inward CFTR-mediated C1- current evoked by
bath perfusion of 3 rn IBMX, 1 nM PGE,, and 1 ~ I M PGF,, in oocytes voltage-clamped to -60 mV. The nucleus of the oocyte was co-injected with 1.4 ng of pcDNAl-hEP, and 2.6 ng of pcDNA-CFTR 48 h before challenge. B, the effect of 3 rn IBMX and 1 PGE, on a n oocyte co-injected with 2.6 ng of pcDNA-CFTR and 1.4 ng of pcDNAcontaining antisense-oriented hEP, cDNA 48 h before challenge. Thearrows indicate the zero current level. The results shown in A and B are representative of 7 and 15 separate experiments,respectively, performed on oocytes from 4 different frogs.
FIG.5. Tissue distribution of hEP, expression. Northern blot analysis of total RNA (10 pgllane) from various human tissuesis shown. A, the blot was hybridized with 3"-labeled hEP, cDNA, as described under "Materials andMethods." B, the amountof total RNA loaded per lane was equal for all human tissues as indicated by the ethidium bromide-stained gel. Positions of 28 S (4.7 kb) and 18 S (1.9 kb) ribosomal RNAs are marked.
However, using reverse transcription-polymerase chain reaction technique, expression was detected in all the above-mentioned tissues including heart, skeletal muscle, retina, and liver, suggesting low level expression of hEP, receptor in these four tissues. The relative levels of expression of the hEP,-encoding mRNAin the restof the tissues examined both by Northern blot analysis or reverse transcription-polymerase chain reaction were comparatively the same (data not shown). re-challenged with 1ILM PGE,. This also resulted in anincrease This distribution is entirely consistent with the previously of inward C1- current which reached saturation a t about 450 nA described putative roles for the EP, subtype in mediating rewithin 7-10 min and reversed to the zero current level upon laxation in the pulmonary, intestinal, renal, and reproductive washing (Fig. 4A).Challenge of oocytes with 1ILM PGF,,, howsystems (2). The high levels of the receptor subtype found in the ever, gave only small current response (20 nA), as expected thymus is of particular importance considering the documented from rank order of potency forthese agonists at the EP receptor effects of PGE, on T lymphocytes. PGE, can have both inhibitory and exitatory effects on T-cells, in some cases inhibiting (2). Similar responses were observed when PGE, and PGF,, were applied in reversed order (not shown), indicating that function and proliferation but also inducing the development of small response to PGF,, was not a result of receptor desensi- mature T-cells fromimmature thymocytes. Atthe present time, tization. In addition, control oocytes co-injected with pcDNA- however, it is difficult to definitively correlate distribution of mRNA with function. The recent study demonstrating coupling CFTR and antisense hEP, cDNA did not respond to PGE, ( 1J~M) despite high levels of CFTR expression, as confirmed by the of bovine EP, isoformsto increases in intracellular CAMP sugeffect of 3 r m IBMX (Fig. 4B). These results show the presence gests that some of the PGE,-mediated events attributed to the of a functional EP, receptor coupled to elevation ofCAMP in EP, subtype may be due to interaction with specific EP, suboocytes injected with pcDNAl-hEP,. type. The availability of specific DNAprobes for the prostanoid Rssue Distribution-Northern blot analysis using total RNA receptors should allow these issues to be resolved. from different tissues revealed that the hEP, gene encodes a Traditionally the distribution and function of the EP, subtype -3.8-kb mRNA and is expressed at some levelin most tissues. has been assigned primarily on the basis of PGE,-mediated Since the cloned cDNAis 1.957 kb long the full-length transcript events that result in anincrease in intracellular CAMPandlor probably contains a considerably longer 5'- andlor 3'-untrans- smooth muscle relaxation. In addition, many studies have used lated sequence. A single 3.8-kb transcript was present in each the putative EP,-selective agonist butaprost to define the EP, of the human tissues examined. The highest expression was receptor. In thisreport, we have cloned a human PGE, receptor found in thesmall intestine, withlower but significant levels in which has high sequence identity with a previously cloned lung, kidney, thymus, uterus, andbrain, and with no expression mouse PGE, receptor (6).The mouse PGE, receptor was desigdetectable in liver, heart, retina, or skeletal muscle (Fig. 5). nated to be the EP, subtype because it displayed reduced affinity
Cloning of the Human Prostaglandin for EP,- and EP,-selective ligands and activation stimulated an increase in intracellular CAMP. To date, the hEP, subtype has similar pharmacological properties to the mousehomologue with, most strikingly, butaprost being essentially inactive at both receptors. Nor is butaprostactive at thehEP, (5)and m P 3 (15) subtypes. Species differences are unlikely to explain this disparity since butaprost hasbeen shownto be active at putative human EP, receptors producing potent inhibition of spontaneous activity in myometrium (27) and relaxation of bronchial muscle (24) in uitro. It isnot possible, therefore, to rule out the existence of another EP receptor, or isoforms of the known EP, subtype, which display high affinity for butaprost. This is supported by preliminary pharmacological evidence showingthat butaprost isless potent at the putative rabbit jugularvein EP, receptor than at the putative cat tracheaEP, preparation (28). It is also possible that thepharmacological actions of butaprost are mediated by a receptor other than the EP receptor. Although recent cloning strategies have greatly advanced our knowledge of EP receptor structure and function, caution must be advised in making a definitive classification until all thevarious receptor subtypes have been identified. In conclusion, the present study reports the cloning and expression of the human prostaglandin E receptor EP, subtype. The signal transduction pathway of the hEP, receptor is coupled to an elevation in intracellular CAMPand the rank order of affinities for the endogenous arachidonic acid-derived prostaglandins and known selective agonists and antagonists is as predicted for the hEP, receptor, except for butaprost, an EP, selective agonist. The cloning of human EP, receptor will allow for detailed structure-function studies, which will provide better understanding of the role for PGE, in normal and disease conditions. Acknowledgments-We thank Barbara Sholzberg for assistance in the preparation of this manuscript. Butaprost and AH6809 were generous gifts from Miles Inc. and Glaxo Group ResearchLtd., respectively, MB28767 was synthesized by Michel Belley from the Department of Medicinal Chemistry at the Merck Frosst Centre for Therapeutic Research. REFERENCES 1. Campbell, W. B. (1990)in The Pharmacological Basis ofTherapeutics (Gilman, A. G., Rall, T. W., Nies, A. S., and Taylor, P., eds) 8th Ed., pp. 600-617, Pergamon Press, New York 2. Coleman, R. A., Kennedy, I., Humphrey, P. P. A,, Bunce, K., and Lumley, P.
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