cDNA Cloning of a Mouse Prostacyclin Receptor - The Journal of ...

THEJOURNAL OF BIOLWICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 13, Issue of April 1, pp. 9986-9992, 1994 Printed in U.S.A.

cDNA Cloning of a Mouse Prostacyclin Receptor MULTIPLE SIGNALING PATHWAYS AND EXPRESSION IN THYMIC MEDULLA* (Received forpublication, October 14, 1993, and in revised form, December 27, 1993)

Tsunehisa NambaSI, Hiroji OidaS, Yukihiko Sugimoton, Akira KakizukaS, Manabu Negishin, Atsushi Ichikawan, and Shuh NarumiyaSII From the Departments of $Pharmacology and $Anesthesia, Faculty of Medicine, and the Wepartment of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan

A functional cDNA for a mouse prostacyclin receptor vating adenylate cyclase and generating CAMP.These actions was isolated from a mouse cDNA library by reverse tran- can counteract the proaggregatory and vasoconstrictor activiscription polymerase chain reaction and hybridization ties of another unstable prostanoid, and contributes to screening.The cDNAencodes a polypeptide of 417 amino themaintenance of homeostasisin circulation(Moncada, acid residues with putative seven transmembrane do- 1982). These activitiesof prostacyclin have been regarded usemains and an calculated molecular weight of 44,722. The ful inpreventingandmanagingdiseasessuch as cerebral amino acid sequence is 3040% identical in the trans- thrombosis and myocardial infarction, and many stable anamembrane domainsto those of the mouse prostaglandin logues of prostacyclin have been synthesized as potential 4receptor. drugs. Using these stable analogues, as well a s prostacyclin (PG) E receptor subtypes and thromboxane [SHIIloprost,a specific prostacyclinreceptorradioliitself, actions of prostacyclin have been extensively studied in gand, specifically bound to the membrane of Chinese platelets, smooth muscles,and other tissues and cells (Coleman hamster ovary cells permanently expressing the cDNA with unla- et al., 1990). These studies have revealed that responses to with Kdof 4.6 m.This binding was displaced beled prostanoids in the order of cicaprost > iloprost, prostacyclin and its analogues are somehow different among both prostacyclinagonists > PGE, > carbacyclin > EPGD, tissues andcells of different species, indicating thepresence of = ST& a thromboxane4agonist f: PGE, > PGF,,. Ilo- the receptor subtypes or coupling of the receptor to more than one species of G protein. It is also suggested that prostacyclin prost in a concentration-dependent fashion increased CAMPlevel and generated inositol phosphates ia these and its agonists cross-react on other types of the prostanoid cells, indicating that the receptor couples to multiple receptors (Dong and Jones, 1982; Armstrong et al., 1989). In signal transduction pathways. Northern blot analysis order to clarify these points, it is essential to elucidate strucrevealed that the mRNA is expressed most abundantly ture, signaling, and distributionof the prostacyclin receptor. in thymus, followed by spleen, heart, and lung. In situ We have thus far determined structures of the human and hybridization of thymus showed thatit is expressed ex- mouse TXA, receptors (Hirata et al., 1991; Namba et al., 1992) clusively in medulla and notin cortex. and three subtypes of the mouse PGE receptor (Sugimotoet a l . , 1992; Hondaet al., 1993; Watabeet al., 1993) by cDNAcloning.

w,

Prostanoidssuchasprostaglandins (PGs),’ thromboxane, and leukotrienes are a family of oxygenated metabolites of arachidonic acidand exerta variety of actions to maintain local homeostasis in the body (Moncada et al., 1985). The actions are mediated by a cell surface receptor specific to each member (Halushka etal., 1989; Coleman etal., 1990).Prostacyclin (PGI,), one of the prostanoids, isa n unstable compound which, in blood vessels, is largely produced by endothelial cells. It inhibits platelet aggregation and inducesvasodilation by acti-

These studiesrevealed that theprostanoid receptors have conserved amino acid sequences and constitute a novel family of the rhodopsin-type receptor superfamily with seven transmembrane domains. We have isolated a cDNA for a prostacyclin receptor by RT-PCR with primers corresponding to the conserved sequences and cDNA screening of a mouse library. Expression studies haverevealed that this receptor can couple t o multiple G proteins to induce both CAMPincrease and phosphoinositide breakdown. A novel function of prostacyclin in immune system is suggested by Northern blot and i n s i t u hybridization analysis.

*This workwassupported in part by Grants-in-aid for Scientific Research 05404020,04255103, 05771975,05671816, and 05454568 from the Ministry of Education, Science, and Culture of Japan and by grants from the Mitsubishi Foundation and the Takeda Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C.Section1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper hasbeen submitted to the GenBankTMIEMBL Data Bank with accession number(s)D26157. 11 To whom corresoondence should be addressed: DeDt.of Pharmacology, Kyoto University Faculty of Medicine, Kyoto 606, Japan. Tel.: 8175-753-4392; Fax: 81-75-753-4693. The abbreviations used are: PG, prostaglandin; G protein, heteroTX, trimeric GTP-binding protein; PCR,polymerasechainreaction; thromboxane; PI, phosphatidylinositol; IP, inositol phosphate;IP,, inositol phosphate; IP,, inositol bisphosphate; IP,, inositol trisphosphate; PBS,phosphate-bufferedsaline; DTT, dithiothreitol; CHO, Chinese hamster ovary; RT, reverse transcriptase; kb, kilobase paids); HBS, HEPES-buffered saline.

EXPERIMENTALPROCEDURES Materials-Iloprost, [3Hliloprost, my0-[2-~H]inositol, [32PldCTP, and ‘251-labeled CAMP assay system were obtained from Amersham Corp. Cicaprost and ST& were generous gifts from Dr. K.-H. Thierauch of Schering (Berlin, Germany) and On0 Pharmaceuticals (Osaka,Japan), respectively.Carbacyclin,PGE,,PGE,,PGF,,, and PGD,were purchasedfromFunakoshi Pharmaceuticals (Tokyo, Japan). Sources of other materials are shown in the text. Molecular Cloning of a Mouse cDNA for a Prostacyclin ReceptorTotal RNA of mouse P-815 cultured mastocytoma cells was isolatedby acid guanidium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987), and the first strand cDNA was synthesized using random primer (Toyobo, Osaka, Japan) and Molony murine leukemia virus reverse transcriptase (Life Technologies, Inc.). Two degenerated PCR primers were synthesized corresponding to the aminoacid sequences highly conserved in second and seventh transmembrane doare CT(G/C)GC(A/ mains of the clonedprostanoidreceptors.These T)GT(G/C)ACNGA(T/C)(T/C)T and A(T/G)(A/G)TANACCCANGG(N

9986

Cloning and Localization of Prostacyclin Receptor

9987

was quickly aspirated and 1 ml of 5% trichloroacetic acid was added. G)TC, corresponding amino to acids LAVTDL a nD dPWM, L3H]IPs formed were separated by Bio-Rad AGlx8 column chromatogrespectively. With the first strand cDNA as a template, amplification in the eluates was performed on a Zymoreactor (Atto, Tokyo, Japan) with twocycles of raphy asdescribed by Bemdge et al. (1983). Radioactivity was determined by scintillation counting using Clear-Sol. 1min of denaturation at 94 "C, 0.5 minof annealing at40 "C, and 1min Northern Blot Analysis-Poly(Al+ RNA was purified using Oligotex of extension a t 72 "C; three cycles of 1min of denaturation at 94 "C, 0.5 dT30 (Takara, Kyoto, Japan) from total RNA prepared from organs of min of annealing at46 "C, and 1min of extension at 72 "C; followed by 27 cycles of 1 min of denaturation at 94 "C, 0.5 min of annealing at 4-5-week-old DDY mice or P-815 cells. Five pg of each poly(Al+RNA 55 "C, and 1 min of extension a t 72 "C. Amplified fragments were sub- was separatedby electrophoresis on a 1.2% agarose gel and transferred cloned into pBluescript SK(+) (Stratagene) and sequenced. A 706-base onto a nylon membrane (Hybond-N, Amersham). 5"Fragmentof CP302 for pair insert namedCY coded an amino acid sequence homologous to the (1.7 kb) was subcloned, linearized withXbaI, and used as a template riboprobe synthesis. AcRNA probe was prepared with [a-s2PlUTP and sequences of the known prostanoid receptors and was used asa probe T7 RNA polymerase (Stratagene). Hybridization was carried out at for plaque hybridization. 70 "C in 5 x SSC, 50% formamide, 5 x Denhardt's solution, 0.2% SDS, A cDNA library of P-815 cells was prepared by an oligo(dT1priming DNA, 200 pg/ml yeast transfer method using acDNA synthesis kit (Pharmacia), size-selectedb 1 . 5 kb), 250 pg/ml heat-denatured salmon sperm x SSC and 0.1% and inserted into the EcoRI site of A-ZAP11 with EcoRI adapters. De- RNA, and 5 ng/ml probe. The filter was washed in 0.1 SDS a t 70 "C for 20 min, treated with 1 pg/ml RNase A in 2x SSC for rivedclones (3.5 x lo5) were transferred t o nylon membranes (Hy10 mina t room temperature, and washed again in 0.1 x SSC and 0.1% bond-N plus, Amersham) and screened by hybridizationCY.toHybridSDS a t 70 "C for 20 min. The filter wasexposed to a Konica x-ray film ization was camed out at 65"C in 6 x SSC containing 5 x Denhardt's (Tokyo, Japan) for 2 days. solution, 0.5% SDS, 200pg/ml heat-denatured salmon spermDNA and I n Situ Hybridization-In situ hybridization of mouse thymus was the radiolabeled probe. Filters were washedtwice a t 65 "C in 2 x SSC camed out essentially as described previously (Shigemotoet al., 1992). containing 1% SDS for 30 min. Nine positive clones were picked and Four- t o five-week-old DDY mice were sacrificedby cervical dislocation. converted to plasmids by the in vivo excision method. Nucleotide seat quences were determinedon both strands with Bca Best DNA sequenc- The thymus was removed immediately and frozen in isopentane ing kit (Takara, Kyoto, Japan). Arepresentativeclone (CP302) was used -70 "C. Sections of 10-pm thickness were cut on a cryostat and thawmounted onto poly-L-lysine-coated slides. They were briefly air-dried for further analysis. and kept at -80 "C until fixation. Thefrozen sections were then warmed Expression in CHO Cells-A stably transformed CHO cell line was established a s follows. A XbaI fragment of CP302 was subcloned into to room temperature and fured with 4% formaldehyde in PBS for 10 0.25% acetic anhydride in 0.1 pEF-BOS (Mizushima and Nagata, 1990) and transfected with pSVbsrmin, rinsed in PBS, and acetylated with M triethanolamine, 0.9% NaCl for 10 min a t room temperature. After carrying a resistant gene for an antibiotic, blasticidin S (Kamakura et al., 1987; suppliedby Funakoshi, Osaka, Japan),a t a ratioof 20 to 1by dehydration in ethanol, the sections were dried and stored at -80 "C until use. Hybridization was carried out in 10 n" Tris-HC1, pH 7.5, lipofection (Felgner et al., 1987). CHO cells were maintained in a-modification of minimal essential medium with 10% fetal calf serum. CHO containing 50% formamide, 2 x SSC, 1 x Denhardt'ssolution, 10% dextran sulfate,0.2% SDS, 100 m~ D m , 500 pg/ml salmon spermDNA, cell clones were establishedby selection with 15 pgiml blasticidin S and and 250 pg/ml yeast tRNA. A riboprobe synthesizedas described above screened by Northern blot analysis.Clones expressing themRNA were using Ia-32SlCTP instead of [a-32PlUTP, was preheated a t 80 "C for 3 used for further analysis. Ligand Binding Assay-The transfected CHO cell clones were cul- min in 1 M Dl", diluted to 1 x lo5 cpm/pl with the hybridizationbuffer, then with cover slips and tured, harvested, and homogenized using a Potter-Elvehjem homog- and added to the sections, which were covered enizer i n a solution containing 25n" Tris-HC1, pH 7.5, 0.25M sucrose, sealed by rubber cement. After incubation a t 57 "C for 5 h, the cover 10 mM MgCI,, 1 m~ EDTA, 0.1 n" phenylmethylsulfonyl fluoride. The slips were removed and the slides were immersed in 2 x SSC, 10 n" homogenate was centrifugedat 800 x g for 10 min. The supernatant was2-mercaptoethanol at room temperature overnight andat 60 "C for 1h. The sections were then treated with 20 pg/ml RNase Ain 0.5M NaC1,lO saved, and the pellet was suspended in the same buffer, homogenized, x SSC at 60 "C for and centrifuged. The supernatants were combined and centrifuged at m~ Tris-HC1, pH 7 . 5 , l n" EDTA, and washed in 0.1 exposed and 100,000 x g for 1h. The pellet was suspended 20 in n" MES, pH6.0,lO 1h. After dehydration in ethanol, the slides were air-dried mM MgCl,, 1 m~ EDTA (the suspension buffer) and used as the crude to pmax film (Amersham)for 2 weeks. membrane. [3HlIloprost binding was examined by incubating 40 pg of protein from the crude membrane in the suspension buffer with various RESULTS concentrations (in Scatchard analysis) or 20 IIM (in displacement experiMolecular Cloning of a cDNA for Prostacyclin Receptorments) [3HIiloprost in a total volume of 100 pI at 30 "C for 1 h. The incubation was terminatedby the addition of 2 ml of the ice-cold sus- Previous studies showed that the amino acid sequences in the pension bufferand the mixture was rapidly filtered through a Whatman transmembrane domains are relatively conserved in various GF/C filter. The filter was then washed four times with 2 ml of the types and subtypes of prostanoid receptors (see, for example, ice-cold suspension buffer, and the radioactivityon the filter was meas- Watabe et al. (1993)).We synthesized degenerated oligonucleured in 5 mlof Clear-Sol scintillation mixture (Nakalai Tesque, Kyoto, otides corresponding tothe conserved sequences in the second Japan). Nonspecific binding was determinedby adding 1000-fold excess of unlabeled iloprost to the incubation mixture.specific The binding was and seventh putative transmembrane domains of other proscalculated by subtraction of the nonspecific binding fromthe totalbind- tanoid receptors, and carried out PCR using these mixed oligoing. nucleotides as primers. We used as a template the first strand CAMPAssay-A CHO clone showing both mRNA expression and cDNA prepared from total RNA of cultured P-815 mouse mas[3Hliloprost binding was seeded at 5 x 104/well in a 24-well dish and tocytoma cells, because these cells express the high level of cultured in the complete medium containing 8 pdml blasticidin S for 72h. Cells were washed with 0.5 ml of Hepes-buffered saline (HBS; 140 prostacyclin receptor(Hashimoto et al., 1990). The PCR amplified multiple length of nucleotides under these conditions. The mM NaCl, 4.7 n" KCl, 2.2 m~ CaCl,, 1.2 n" KH,PO,, 11 m~ glucose, and 15n" Hepes-NaOH, pH 7.4) and preincubated for 10 min in 450 pl products were subcloned into pBluescript, and their DNA seof the solution containing 1 m~ 3-isobutyl-1-methylxanthine at 37 "C. quences were determined. One ofthe fragments, 706 base pairs Iloprost in 50 plof the samesolution wasthen added and incubated for long, showed significant homology with other known prosta10 minat 37 "C. The reaction was terminated by the additionof 500 pl noid receptor cDNA. This fragment, designated CY, was used as of 10% trichloroacetic acid, and CAMP formed was determined by raa probefor plaque hybridization screening in a P-815 cDNA dioimmunoassay using CAMP assay kit. Measurement of Formation of PHIIPs-A CHO cell line permanently library. By this procedure, nine positive clones were isolated. expressing the cDNA was seeded at 2.5 x 105/well in six-well dishes, Restrictionmapping and sequence analysis of these clones cultured for 72 h, and then labeled with 1pCi/ml my0-[2-~H]inositolfor showed that they derived from the same sequence and over24 h as described by Negishi et al. (1990). Where indicated, 20 ng/ml lapped each other. Fig. 1 shows nucleotide and deduced amino pertussis toxin or 4 pg/ml cholera toxin was added to the medium acid sequences of one of these clones, CP302. It contains the during the 24 of h incubation. Cells were washed three times with HBS 3.3-kb insert, which has an open reading frame of 1,251 base and preincubated in 2 mlof HBS containing 10mM LiCl for 10 min at pairs. Deduced amino acid sequence shows that it encodes a 37 "C. The solution was aspirated, and the reaction was started by adding 1ml of HBS containing 10n" LiCl and various concentrations polypeptide of 417 amino acids with a calculated molecular of iloprost. After incubation at 37 "C for indicated times, the medium weight of 44,722. Hydrophobicityprofiledetermined by the

9988


:27 :200 :67 :320 : 107

:440 : 147

:560 :187 :680

:227 :800 :267 :920 :307 :1040 :347 :1160 :387 :1280 :417

:1400 :1520 :1640 :1760 :1880 :2000

:2120 :2240 :2360 :2480 :2500 :2720 :2840 :2960 :2995

FIG.1. Nucleotide and deduced amino acid sequences of the mouse prostacyclin receptor cDNA. The nucleotide sequence is numbered in accordance with the first methionine of the longest open reading frame. The deduced amino acid sequence is shown beneath the nucleotide sequence in single-letter code. Positions of the seven transmembrane domains (I-VII) were estimated on the basis of a hydrophobicity profileand sequence comparison with other G protein-coupledreceptors, and are indicated by solid burs. *, potential N-glycosylation sites in the extracellular region; +, potential phosphorylation site by CAMP-dependent protein kinase; #, potential phosphorylation sites by C-kinase.

Kyte and Doolittle method (Kyte and Doolittle, 1982) and the sequence homology analysis indicate that it possesses seven hydrophobic segments, suggestingthat it is a rhodopsin-type G protein-coupled receptor. The amino acid sequence showed 39, 32, 28, and 32% identity in the transmembrane domains to those of EP2, EP3, and EP1 subtypes of prostaglandin E and TXA,receptors, respectively. Two potential A m glycosylation sites (Bause, 1983) are assigned in the N-terminalregion and the first extracellularloop as indicated by asterisks in Fig. 1.A potential phosphorylation site by CAMP-dependent protein kinase is found in the first cytoplasmic loop (Glass et al., 1986) and thoseby protein kinase C are found in the thirdcytoplasmic loop and the C-terminal tail (Woodgett et al., 1986) (Fig. 1). Because we have found that the isoforms of bovine PGE receptor subtypeEP3 are createdby alternative splicing at the C-terminal tail and that theycouple to different signal transduction cascades (Namba et al., 19931, we have examined the sequences of all thepositive clones obtained. However, we could not find isoforms created by alternative splicing. Characterization of Ligand Binding Properties and Signal for this receptor, we Dansduction-To identifytheligand transfected CHO cells with CP302 and isolated seven inde-

pendent clones permanently expressingits mRNA. All of these clones showed specific binding of [3Hliloprost, a specific radioligand for the prostacyclin receptor (Town et a l . , 1982),while no specific binding wasfound either in untransfectedCHO cells or in mock-transfected cells (data not shown). Scatchard analysis in one of the clones with [3H]iloprost yielded a dissociation constant of 4.5 m and maximal binding of 4.6 pmoYmg protein. Fig. 2 shows the specificity of this binding. Specific [3Hliloprost binding wasdisplaced in a concentration-dependent mannerby various unlabeled prostanoids in the order of cicaprost > iloprost, both prostacyclin agonists > PGE, > carbacyclin, a prostacyclin agonist >> PGD, = ST&, a thromboxane 4 agonist = PGE, > PGF,,. This orderis quite similar to the pharmacologically defined rank order of potency of these compounds on the prostacyclin receptor in various tissues (Armstrong et al., 1989; Coleman et al., 1990),and theaffinity and specificity of L3H]iloprost binding in the transformantis almost identical to those obtained for the prostacyclin receptor in other tissues and cells (MacDermot et al., 1981; Town et al., 1982; Leigh et al., 1984; Tsai et al., 1989; Hashimoto et a l . , 1990). We, therefore, conclude that the receptor encoded by CP302 is a prostacyclin receptor.

Localization Cloning and 100

40

20

;

Ligand Concentration(-log M)

of

Prostacyclin Receptor

9989

20

I

IA

’ 0

FIG.2. Displacement of [3Hliloprostbinding in membranes of CHO cells stably transformed with CP302.Membrane of CHO cells transfected with CP302 was incubated with 20 nM of [3Hliloprost and various unlabeled prostanoidsof indicated concentrations. [3HlIloprost binding was assayed as described under “Experimental Procedures.” Compounds used were iloprost(m),cicaprost (O),carbacyclin (01,PGE, (O), PGE, (+), PGD2 (A),ST& (A),and PGF, (VI.

2

4

6

8

1

0

time (mh)

300

E

250

-

6 200 0

200

+/

I

I

150

I

E

E’ 0 n

-

p!

loo

I

I

I

I

50

t lloprost Concentration(-log M)

350

l

A

C

0

300 0

5 lloprost Concentration (-log M )

200

E, 150

B

FIG.3. Effect of iloprost on C A M P level in CHO cells expressing 100 the receptor. The same CHO cells as inFig. 2. were incubated with the 1 2 3 indicated concentrations of iloprost in the presence of 1 m~ 3-isobutyl1-methylxanthine, andcAMP level was determined as described under FIG.4. Effect of iloprost on the phosphoinositide metabolism. “Experimental Procedures.” The results shown are the mean 2 S.E. for A, time course of the formation of [3HlIPs. CHO cells were prelabeled triplicated determinations. and incubated with 1 p~ iloprost for the time indicated. The reaction was terminated and IPS were separated as described under “ExperiAs shown in Fig. 3, iloprost generated CAMPin these cells in mental Procedures.”0, [,H]IP,; 0, [3HlIP,; 0,L3H1IP,. B , concentration a concentration-dependent fashion. The increase was detecta- dependence of IP, formation. Cells were incubated with indicatedconof iloprost for 2 min, and [3HlIP, was separated and measble at 10 PM and reached plateau a t 1 nM, giving the EC,, value centration ured. C , cells were pretreated with (column 2 ) or without (columns 1 of -100 PM. Other CHO cell clones, which displayed specific and 3 ) F T for 12 h and treated with1 PM iloprost (columns 1 and 2 ) or iloprostbinding,also generated CAMP to iloprost, while no 1 mM dibutyryl cAMP for 2 min. [3HlIP, was separated and measured. The values of IPS are means f S.E. of three determinations. response was found in untransfected CHO cells.

In addition to the CAMP response, we have found that iloprost evokes PI breakdown in thecells expressing the receptor. Fig. 4A shows the time courses of the formation of PI metabolites. L3H1IP3formation was observed at 30 s after the addition of 1p~ iloprost and reached the maximum level of about %fold of the control at 2 min. [3HlIP, formation could also be detected at 30 s and increased continuously over a 10-min period. In contrast, L3H1IP, accumulation was detected only after 2 min and then increased linearly. These time courses correlate well t o those found in thetypical PI metabolism in CHO cells evoked by such agonists as tachykinins (Nakajima et al., 1992).This PI response took place in a concentration-dependent manner, as shown for [,HIIP3 formation in Fig. 4B; the EC,, value for iloprost was 100 nM. The response was insensitive to pertussis toxin pretreatment and wasnot evoked by the addition of 1mM dibutyryl CAMP,as shown in Fig. 4C. To exclude the possibility that G, or its p y subunits mediate both CAMPgeneration and PI response, we incubated theCHO cells with cholera toxinfor 24 h to degrade G, as described by Chang and Bourne (1989).

While this procedure eliminated the iloprost-induced cAMP generation, it did not affect the PI response evoked by this compound (data not shown). These results suggests the receptor couples to multiple G proteins, G, and G,. Tissue Distribution of the Prostacyclin Receptor-The expression of the prostacyclin receptor in various mouse tissues and P-815 mastocytoma cells was examined by Northern blotting (Fig. 5). The mouse prostacyclin receptor mRNA seems to be a single species of 3.5 kb, which is expressed in several tissues hut most abundantly in thymus, followed by spleen, heart, and lung in this order. The expression in other tissues was under detectable level in this condition. RT-PCR using primers specific to CP302 with mRNAs prepared from these tissues and the P-815 cells amplified the fragment of the expected size, suggesting the absence of alternatively spliced mRNA in these tissues (data not shown). The mRNA expression in thymuswas further examined by in situ hybridization (Fig. 6). Positive signal was visible only in


9990 1 -

2

3

4

-

5

6

7

8

9 1 0 1 1 1 2

A

28s-

c

FIG.5. N o r t h e r n blot analysis of various m o u s e organs. Poly(A)+ RNAs were isolated from the respective mouse organs, and5 pg of RNA wasappliedineachlane.Hybridizationwascarriedoutusing :12Plabeled invitro transcribed antisense RNA a s a probe a s described under “Experimental Procedures.” left A arrow indicates the bandscorresponding to the mRNA. Lane 1 , brain; lane 2, thymus; lane 3, lung; lane 4 , heart; lane 5,liver; lane 6,stomach; lane 7, spleen; lane 8,ileum; lane 9,kidney; lane 10, testis; lane 11, uterus; lane 12, P-815.

medulla and not in cortex. As shown in Fig. 6C, the signal disappeared by the addition of the excess amount of cold cRNA.

DISCUSSION Ligand Receptor Interaction Suggested by Sequence Comparison with other ProstanoidReceptors-In rhodopsin-type receptors, the ligand binding isbelieved to be determined by the transmembrane domains (O’Dowd et al., 1989). Fig. 7 shows the amino acid sequence homology between the prostacyclin receptor and the otherprostanoid receptors. There are several conserved amino acids in the transmembraneregions. Some of them are also conserved in otherrhodopsin-type receptors such as catecholamine andadenosine receptors, suggestingthat they are important in forming receptor structure or interaction with a G protein. The amino acids that are conserved in the prostanoids receptors but not in otherrhodopsin type receptors may be important in recognizing the structure of prostanoids. These areTyr-105 in thesecond transmembrane domain, Phe132 in the third transmembrane domain, Tyr-218 in the fifth transmembrane domain and Arg-308 and Asp-317 in the seventhtransmembrane domain. PGsand TXs have common structural features such as an a-carboxyl group, a hydroxyl group at position 15, and two aliphatic side chains. These structures are believed to play an important role in interaction between the prostanoid and itsreceptor because a modification of these structures results in dramatic decrease binding in affinity (Coleman et al., 1990). The conserved amino acids noted above may be important for interaction with these ligand structures. The structure activity relationship of prostacyclin has been studied intensively (Tsai and Wu, 1989). It suggests important structuresspecific to prostacyclin. Amino acids that are present in this receptor but not conserved in other prostanoid receptors (such as Phe-96, -176, -307, -309, -312, -321 and -324; Thr-124, -137 and -227; Cys-95 and -177; and Asn310) might be important in recognizing these structures. We believe that assignment of ligand binding domain in the receptor structure will be possible on the basis of the sequence information discussed above and by introducing mutations to presumed critical amino acids of the receptor. Such studies will help designing specific agonists and antagonists notonly for prostacyclin but for other prostanoid receptors. Prostacyclin Receptor MediatesMultiple Signaling-The findings in various systems suggest that prostacyclin stimulates adenylate cyclase and produces CAMP(Halushka et al., 1989). Here we have verified this proposal by expressing the cloned receptor and examiningCAMPresponse. The EC,, value

FIG.6. Distribution of the prostacyclin receptor mRNA in mouse thymus. Thymus sect~ons from 4-5-week-old DDY strain mice werehybridizedwith anantisense rihoprohe correspondingto the 1.7-kb fragment of 5 ’ end of CP302 in the absence ( B )or presence ( C ) of 100-fold excess of unlabeled probe. A, hematoxylin-eosin staining section. B and C, autoradiogram. A C represent adjacent sections. Cx, cortex; Med, Medulla; bar, 2 mm.

of the response for iloprost in the transformant is10 times less than its Kd value, while the EC,, value was equal to theKd in P-815 mastocytoma cells (Negishi et al., 1991), suggesting the receptor-effector coupling is more efficient in CHO cells than in the mastocytoma cells. Such variations in coupling efficacy of the prostacyclin receptor was observed in platelets of various species (Armstrong et al., 1989). In addition to CAMPgeneration, we identified that this receptor mediates PI breakdown. Some researchers report that prostacyclin or its analogues evoke signaling pathways other than CAMP generation. For example they cause smooth muscle contraction in rabbit stomach (Whittleet al., 1979) and guineapig ileum (Lawrence etal., 1992) and [Ca2+l,increase in several lines of cultured cells (Watanabe et ul., 1991; Schwaner et ul., 1992; Vassaux et ul., 1992). However, it has remained tobe determined whether these responses are mediated by prostacyclin receptor or by cross-reaction of the agonistson different types of prostanoid receptors such as EP1 (Dong and Jones, 1982; Armstrong et al., 1989). Thepresentstudy shows the prostacyclin receptor indeed couples to a G protein other than G,. Such multiplicity in signaling pathwaysmay be important in some of the prostacyclin

Localization Cloning and IP EP2 EP3 EP 1 TP

IP EP2 EP3 EP1 TP

of Prostacyclin Receptor

I MKMMASDGHPGPPSVTPGSPLSAGGREWQGMAGSCWNITYVQDSVG MAEVGGTIPRSNRELQRCVLLTTTIMSIPGVNASFSSTPERLNSPVTIPAVMFIF MASMWAPEHSAEAHSNLSSTTDDCGSVSVAFPITMMVT MSPCGLNLSLADEAATCATPRLPNTSVVLPTGDNGTSPALPIFSMTL MWPNGTSLGACFRPVNITLQERRAIASPWFAASFCAL

LLVSPVTIAT ; I ; S + ~ ~ S L L GQW-----PGDQ G L A H G G T F $ ~ T C Y111 VIPG LVTGAI

9991 II A""RRRSHPSAFA K--SRKEQKETTFYTRSYRRRESKRKKSF-QVAGRMRRRRSAATFLLFVAS-

GARPGAGPRSSFLA---DCG-FYSHYVDK-IV

WYASHMKTSQRRWEQLDPSGR C F AGRA-PAGGA----FLGG S Q W L D W R A T D P S C R - C FMGV

RL

VLAAMALAV

LVWVAAGAL

IP EP2 EP3 EP1 TP

VI

EP2

GQ :193 ER :188 sv :177 EL :183 sv :174

:247 :280 :243 :275 :226

IP EP2 EP3 EP 1 TP

IP EP2 EP3 EP TP

:94 :94 :90 :90 :77

VI1 TQA-----IAPDSREMGD-----INQLYQPNVVKDISRNPD-----KMIFNQMSVEQCKTQMGKEKECNS VVLAIGGWN-SNSLQ--R-PL--QTLLQTPPVMSFSGQLLRATE-HQ

:329

:368

:332 :365

:315

:417 -QRLKFWLCCLCARSVHGDLQAPLSRPASGRRDPPAPTSLQ~EGS~PLSSWGTGQVAPLTAVPLTGGDGCSVG~SKSEAIAACSLC IEKIKCLFCRIGGSGRDSSAQHCSESRRTSSAMSGHSRSF~LKEISSTSQTLLYLPDLTESSLGGRNLLPGSHGMGLTQADTTSLRTLRISETSDSS :468 :365 FCQIRDHTNYASSSTSLPCPGSSALWSDQLER :405 1LRLLPLRVSAKGGPTELGLTKSAWEASSLRSSRHSGFSHL :341 -------LHPRFSSQLQAVSLRRPPAQAMLSGP

-"""

QGQDSESVLLVDEVSGSHREEPASKGNSLQVTFPSETLKLSEKCI

:513

FIG.7. Comparison of the amino acid sequences among types and subtypes of prostanoid receptors.The amino acid sequences of the prostacyclin receptor (ZP), EP2, EP3, and EP1 subtypes of PGE, receptors and ?xA, receptor (TP)are aligned to achieve the maximal homology using a computer program. Amino acids conserved in at least four receptors are indicated by boxes. Dashes show deletions of amino acids when compared with other sequences. Positions of putative transmembrane domains were indicated as Z-VZZ.

actions (Vassaux et al., 1992). Because there is a difference in potencies of prostacyclin analogues in some tissues and cells, some investigators have proposed that receptor subtypes exist for prostacyclin (Cornishi et al., 1989). However, our screening of the cDNA library of P-815 cells and RT-PCR study in various mouse tissues revealed no homologous or alternatively spliced mRNA, and Southern blot analysis of mouse genome revealed only single bands corresponding to the cDNA, excluding the presence of the duplicated or highly homologous genes.' Thus, we conclude that other subtypesor isoforms of prostacyclin receptor do not exist in thesecells and tissues examined, although we can not exclude the possibility of the presence of less homologous subtypes or isoforms in other cells or tissues. These findings together with our examination in its signaling pathway suggest the difference so far reportedis derived from the coupling with different G proteins. Expression of Prostacyclin Receptor in ThymicMedulla-The highest mRNA expression was observed in thymic medulla where CD4' or CD8' single positive thymocytes harbor. We detected a statistically significant amount of specific [3H]iloprost binding and generation of CAMP to iloprost in thymocyte suspension in our preliminary experiment^.^ These observations suggest that the receptor is expressed in single positive thymocytes. Several groups reported the prostacyclin producT. Namba and S. Narumiya, unpublished observation. T. Namba, H. Oida, Y. Sugimoto, A. Kakizuka, M. Negishi, A. Ichikawa, and S. Narumiya, unpublished observation.

tion in thymus. They found that, whereas the cyclooxygenase activity in lymphocytes including thymocytesis negligible (Goldyne, 19881, isolated fractions of thymic nurse cells and phagocytic cells produce large amountsof prostacyclin (McCormack et al., 1991; Homo-Delarche et al., 1985). Prostacyclin, therefore, seems tobe produced by thymic stromal cells and to act on mature thymocytes. Interestingly, we have recently found that TXA,receptor is also expressed abundantly in thymus, especially in CD4/CD8 double-negative and double-positive immature thymocytes, a n d t h a t S Tinduces 4 apoptosisin thesecells (Namba et al., 1992; Ushikubi et al., 1993). It is likely that prostacyclin and TXA,work on thymocytes at different stages and regulate their maturation anddifferentiation. Expression of Prostacyclin Receptor in Vascular SystemVasodilator actions of prostacyclin is well known (Coleman et al., 1990). The mRNA of the receptor is expressed abundantly in heart and lung which are rich in vasculature.However, only small amounts of mRNA are detected in other vessel-rich organs such as liver and kidney. This suggests that thereceptor density differs among the vessels or that the cells other than vessels express high amount of mRNA in heart and lung. We are now examining the precise localization of the receptor in these organs by in situ hybridization. Acknowledgments-We thank Prof. K. Mori and N. Mizuno for encouragement, Drs. F. Ushikubi, M. Hirata, R. Shigemoto, T. Ishii, and H. Toh for useful discussions, M. Oka for technical assistance, and Dr. K.-H. Thierauch of Schering and Ono Pharmaceuticals for providing Cicaprost and ST&, respectively.

9992

Localization Cloning and

of Prostacyclin Receptor

Mizushima, S., and Nagata, S. (1990)Nucleic Acids Res. 18, 5322 Moncada, S. (1982)Br J . Pharmacol. 76, 3-31 Armstrong, R. A,, Lawrence,R. A,, Jones,R. L., Wilson, N. H., andCollier, A. (1989) Moncada, S., Flower, R. J., and Vane, J. R. (1985)The Pharmacological Basis of Therapeutics, 7th Ed., pp. 6604573,Macmillan Publishing, New York Br J . Pharmacol. 97,6574568 Nakajima, Y., Tsuchida, K., Negishi, M., Ito, S., and Nakanishi, S. (1992)J. Biol. Bause, E. (1983)Biachem. J. 209,331-336 Bemdge, M. J., Dawson, R. M. C., Downes, C. P., Heslop, J. P., and Imine, R. F. Chem. 267,2437-2442 (1983)Biochem. J. 212, 473482 Namha, T., Sugimoto, Y., Hirata, M., Hayashi, Y., Honda, Y., Watabe, A,, Negishi, Chang, F., and Bourne, H. R. (1989)J. B i d . Chem. 264,5352-5357 M., Ichikawa, A,, and Narumiya, S. (1992)Biachem. Biophys. Res. Commun. Chomczynski, P., and Sacchi, N. (1987)Anal. Biochem. 162,156159 184, 1197-1203 Coleman, R. A., Kennedy, I., Humphry, P. P. A,, Bunce,K., and Lumley, P. (1990)in Namba, T., Sugimoto, Y., Negishi, M., Irie, A,, Ushikubi, E , Kakizuka, A,, Ito, S., Comprehensive Medicinal Chemistry,pp. 643-714,Pergamon Press, Oxford Ichikawa, A., and Narumiya, S. (1993)Nature 365, 166170 Corsini,A., Folco, G. C., Fumagalli,R., Nicosia, S., NoB, M.A., and Oliva,D. (1987) Negishi, M., Hashimoto, H., Yatsunami, K., Kurozumi, S., and Ichikawa, A. (1991) Br J. Pharmacol. 90,255-261 Prostaglandins 42,225237 Dong, Y. J., and Jones, R. L. (1982)Br J . Pharmacol. 76, 149-155 Negishi, M., Ito, S., and Hayaishi, 0.(1990)J. Biol. Chem. 265,61824188 Felgner, P. L., Gadek, T. R., Holm,M., Roman, R., Chan, H. W., Wenz, M., Northrop, ODowd, B. F., Lefkowitz, R. J., and Caron, M. G. (1989)Annu. Reu. Neurol. 12, J . P., Ringold, G. M., and Danielsen, M. (1987)Proc. Natl. Acad. Sei.U. S . A. 84, 67-83 7413-7417 Schwaner, I., Seifert, R., and Schultz, G. (1992)Biochem. J. 281,301-307 Glass, D.B., El-Maghrabi, M. R., and Pilkis, S. J . (1986)J . B i d . Chem. 261, Shigemoto, R., Nakanishi, S., and Mizuno, N. (1992)J. Comp. Neurol. 322, 1212987-2993 135 Goldyne, M. E. (1988)Prog. Allergy 44, 140-152 Sugimoto, Y., Namba, T., Honda, A,, Hayashi, Y., Negishi, M., Ichikawa, A., and Halushka, P. V.,Mais, D. E., Mayeux, P. R., and Morinelli, T. A. (1989)Annu.Reu. Narumiya, S. (1992)J. Biol. Chem. 267,64634466 Pharmacol. Toxicol. 10, 213-239 Town, M.-H., Schillinger, E., Speckenbach,A,, and Prior, G. (1982)Prostaglandins Hashimoto, H., Negishi, M., and Ichikawa, A. (1990)Prostaglandins 40,491-505 24,61-72 Hirata, M., Hayashi, Y., Ushikubi, F., Yokota, Y., Kageyama, R., Nakanishi, S.,and Tsai, A.L, Hsu,M . J . , ViGeswarapu, H., and Wu, K. K. (1989)J . Biol. Chem. 264, Narumiya, S. (1991)Nature 349, 617420 6147 Homo-Delarche, F., Duval, D., and Papiernik, M. (1985)J. Immunol. 135,506512 Tsai, A,-I., and Wu, K. K. (1989)Eicosanoids 2, 131-143 Honda, A,, Sugimoto,Y., Namba, T., Watabe, A., Irie,A., Negishi,M., Narumiya, S., Ushikubi, F., Aiba, Y., Nakamura, K., Namba, T., Hirata, M., Mazda, O., Katsura, and Ichikawa, A. (1993)J. Biol. Chem. 268, 7759-7762 Y., and Narumiya, S. (1993)J. Exp. Med. 178, 1825-1830 Kamakura, T., Kobayashi, K., Tanaka, T., Yamaguchi, I., and Endo,T. (1987)Agric. Vassaw, G., Gaillard, D., Ailhaud, G., and Negrel, R. (1992)J. B i d . Chem. 267, B i d . Chem. 51,3165-3168 11092-11097 Kyte, J., and Doolittle, R. F. (1982)J. Mol. Biol. 157, 105132 Lawrence, R. A,, Jones, R. L., and Wilson, N. H. (1992)Br. J. Pharmocol. 105, Watabe, A,, Sugimoto, Y., Honda, A,, h e , A,, Namba, T.,Negishi, M., Ito, S., Narumiya, S., and Ichikawa, A. (1993)J. Biol. Chem. 268, 20175-20178 271-278 Leigh, P. J., Cramp, W. A,, and MacDermot, J. (1984)J. B i d . Chem. 259, 12431- Watanabe, T., Yatomi, Y., Sunaga, S., Miki, I., Ishii,A., Nakao, A., Higashihara, M., Seyama, Y., Ogura, M., Saito, H., Kurokawa, K., and Shimizu, T. (1991)Blood 12436 78, 2328-2336 MacDermot, J., Barnes, P. J., Waddell, K. A,, Dollery, C. T., and Blair, I. A. (1981) Whittle, B. J. R., Mugridge, K. G., and Moncada, S. (1979)Eur J . Pharrnacol. 53, Eur J. Pharmacol. 75, 127-130 167-172 McCormack, J. E., Kappler,J., Marrack, P., and Westcott,J . Y. (1991)J. Immunol. Woodgett, J . R., Gould, K. L., and Hunter, T.(1986)Eur J. Biachem. 161, 177-184 146,239-243

REFERENCES