Molecular Cloning and Expression of a cDNA of the Bovine ...

Vol. 269, No.5 , Issue of February 4, pp. 3881-3886, 1994 Printed in U.S.A.

hJOURNALOF B ~ O ~ I C A HL EM~Y 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular Cloning and Expressionof a cDNA of the Bovine Prostaglandin Fza Receptor* (Received for publication, August 16, 1993)

Kazuichi Sakamoto, Toshihiko Ezashi, Keiko Miwa, Emiko Okuda-Ashitaka, Takeshi HoutaniS, Tetsuo SugimotoS, Seiji Itos, and Osamu Hayaishi From the Department of Cell Biology, Osaka Bioscience Institute, Suita 565, Japan andthe $Department of Anatomy, Kansai Medical University, Moriguchi 570, J a p a n

termed “inn” was revealed to be PGF2, (41,it is well known Capitalizing on thesignificantsequencehomology that PGF2, causes smooth muscle contractionof uterus, broncomprising the transmembrane motif regions known of prostanoid receptor family, we targeted the cloning ofchus a and trachea, gastrointestinal tract, and blood vessels (1, cDNA clone for prostaglandin(PG) Fa,, receptor from a 5). It has also been recognized for over two decades that PGF2, bovine corpus luteum cDNA library. By using several is of prime importance in initiating luteal regression in many pairs of degenerated primers created from a common species mo(6).PGF2, induces DNAsynthesis and cell proliferation tif of transmembrane domains, polymerase chain reac- in quiescent Swiss3T3 cells (7, 8 ) a n dinvolves pain transmistion gave a clone SN463 carrying the homologous se- sion in the central nervous system (9). T h u s PGF2, produces a quence, which covered transmembrane motif IV-VI of broad range of biological actions in diversetissues through its thethromboxane (“X) 4 receptor.Thispolymerase binding to specific receptors on plasma membranes (1,5 ) . Alchain reaction product was used aa a DNA probe for the though signal transduction of PGF2, through Ca2+ mobilization following cross-hybridization, and a clone BC2211 carvia an increase in phosphoinositide metabolism has recently rying a 2.2-kilobase pairDNA insert was isolated. This clone encodes a protein of 362 aminoacid residues(M,= been reported in many cell types including luteal cells(8, 10161, little is known about the molecular structure of the PGF2, 40,983) with seven potential transmembrane domains abundant receptors for PGF2a, and represented significant overall sequence homologyreceptor. Corpora lutea have sites have been and virtually all studies into PGFz,-binding Into humanTXA,receptor protein (34% in amino acid). jection of the mRNAsynthesized invitro from the cloned conducted with corpora lutea of various species (17-20). Recently cDNAs for prostanoid receptors, human thromboxane cDNAintoaxenopus oocyte elicited electrophysiological (“X) A2 receptor (211, and mouse PGE receptor subtypes EP3, response to PGFa,. Ligand binding displacement in membranes of mammalian COS-7cells transfected with the EP2, and EP1 (22-24) have been cloned successively. To elucidate the structure and function of PGF2, receptors, we have cDNA indicated the rank order of affinity of the receptor to PGs: PGF2m> PGD2 > PGE2 > ST&, a TXAa agonist. used a polymerase chain reaction (PCR) approach to isolate a PGFzu activated inositol phosphate formation COS-7 in cDNA encodingthe PGFZareceptor froma bovine corpus luteal cells transfectedwithreceptor cDNA.Northernblot cDNA library. We report here the complete amino acid sequence analysis and in s i t u hybridization indicated that the of the bovine PGF2, receptor deduced from the cloned cDNA, PGF2,, receptor mRNA is highly expressed and accumu-the electrophysiological and pharmacological characterization lated in corpus luteum. This is the first reporton a suc- of this receptor expressed in Xenopus oocytes and in mammacessful cloningof functional receptor cDNA for PGF2,,. lian cells, and expression patternof t h e PGF2, receptor.

.

EXPERIMENTALPROCEDURES Prostaglandin (PG)’F2,, along with PGE2, is among the first Materials-Materials were obtained from the following sources.The PGs to be isolated in the early 1960s and referred to as the TAcloningkit was from Invitrogen, the mRNA purification kit was from “primary PG”(1).Since a smooth muscle stimulating substancePharmacia LKB Biotechnology Inc., Superscript was from Life Techextracted from swineand sheep lungs (2,3) and from rabbitiris nologies, Inc., nitrocellulose membrane filters BA-S85 and nylon membrane filters NYTRAN were from Schleicher & Schuell, Taq DyeDeoxy * This work was supported in part by grants-in-aid for scientific re- terminator cycle sequencing kit was from Applied Biosystems; rnyosearch on priority area, for scientific research (C) (04670170), and for [2-3Hlinosi~l(20.0 CUmmol) and Megaprime DNA labeling system (168 Ci/ encouragement of young scientists (05770108) from the Ministry of werefrom Amersham Corp., [5,6,8,9,11,12,14,15-3HlPGF~a Education, Science and Culture of Japan, and by grants from the Min- mmol) was from DuPont NEN, and alkaline phosphatase-conjugated istry of Health and Welfare of Japan, Yamanouchi Foundation for Re- anti-digoxigenin antibody was from Boehringer Mannheim. Unlabeled 9a,llp-PGF2, and 9,11-epithio-11,12-methano search on Metabolic Disorders, the Sasakawa Health Science Founda- PGD,,PGE,,PGF,,, X A , (STA,) were generous giRs from Ono Pharmaceuticals, Osaka, tion, the Osaka Gas GroupWelfare Foundation, and the Naito T Foundation. The costs of publication of this article were defrayedin part Japan. Cicaprost was kindly provided by Dr. K.-H.Thierauch, Schering by the payment of page charges. This article must therefore be hereby AG, Germany. All other chemicals were of reagent grade. marked “advertisement” in accordance with 18 U.S.C.Section1734 Designing of Degenerated Primers for PCR-For the PCR screening, solely to indicate this fact. degenerated primers comprising the common motif of transmembrane The nucleotide sequence($ reportedin this paper hasbeen submitted domains 111, IV, VI, and VI1 were designed, and these oligonucleotides to the &nBankmIEMBL Data Bank with accession nurnber(s) 017395. were synthesized by a DNA synthesizer model 381A (Applied Biosys0 To whomcorrespondence should be addressed: 6 2 - 4 Furuedai, tems, Inc.). The primer sequences were derived from the most homoloSuita 565, Japan. The abbreviations used are: PG, prostaglandin; “X,thromboxane; gous regions of the transmembrane domain or neighboring common PCR,polymerase chain reaction; STA,, 9,11-epithio-11,12-methano motif of mouse EPI (24), EP2 (23), EP3 (221, bovine EP3 (25), bovine X A , (21) receptors. The sequences of oligonucleoTXA,; InsP, inositol phosphate; PIPES, piperazine-N,l\r”bis(2-ethane- TXA2,’ and human T sulfonic acid); InsP1, inositol monophosphate; InsP,, inositol bisphos- tides employed for PCR were shown as follows: sense primers, DGPIII, phate; InsP,, inositol trisphosphate; TD, transmembrane domain; bp, base paifis). K. Sakamoto, unpublished data.

3881

Cloning of the Bovine PGF,, Receptor

3882

(GC)(TC)G(GC)CATG(GA)(GC)C(ATG)(TC)(AGC)GAGCG; DGPIV, CA(AG)T(AG)(GC)CC(GTC)GG(GC)(AT)C(GTC)TGGTG(TC)'IT, antisense primers, DGPVIA, T(GC)(GC)AGCAGAT(GC)A(AG)C(AG)(AC) CACCA; DGPVIB, (AC)(AC)CAT(AG)AT(GC)CCCAG(GT)AGCTG; DGPVIIA, ATCCTGGACCC(CA)TGG(AG)T(GT)TACATCCT; DGPVIIB, TGGC(CT)(AT)C(AGC)(TCNGT)GAACCAGAT(TC)CT.

standardmediumstringencyas follows:50% (v/v) formamide/O.l% SDS/6 x standard saline citrate (SSCY20 m~ sodium phosphate, pH 7.0/1 x Denhardt's solution/lO% (w/v) dextran sulfate/sonicated salmon sperm DNA at 100 pg/ml. Hybridization was done a t 40 "C, and the membrane was washed with 1 x SSC and 0.1% SDS a t 42 "C. In SituHybridization-Sense or antisense riboprobe RNA was transcribed in vitro from BamHI- or EcoRV-digested BC2211 DNA in the presence of digoxigenin-11 UTP. Tissue blocks of bovine corpus luteum were fixed with 4% paraformaldehyde and0.12 M sodium phosphate (pH 7.3) and cut into frozen sections of 30-pm thickness. The free floating tissue sections were hybridized with digoxigenin-labeled riboprobe in the following solution a t 45 "C: 50% formamide, 4% dextran sulfate,250 pg/ml salmon sperm DNA, 250 pg/ml yeast RNA, 1 x Denhardt's solution, 0.2% SDS, 0.75 M NaCI, 25 m~ PIPES. Hybridized sections were then washed with 0.1 x SSC after the treatment with RNase Afor 30 min a t 37 "C. The hybridization material was reacted with alkaline phosphatase-conjugated anti-digoxigenin antibody and then detected by color reaction in the incubation medium containing nitroblue tetra-

PCR Amplification-Amplification was performed using a combination of possible pair of sense and antisense primers.Amplification reaction was performed under the low annealing conditions a s follows: 95 "C for 1 min, 48 "C for 2 min, and 72"C for 2 min for 25 cycles. The PCR products were directly cloned into PCR-I1 vector DNA with a TA cloning kit, and theclones were screened by blue-white selection.Positive clones that carry the insert with an expected size were directly sequenced by a DNA sequence analyzer model 737-A (Applied Biosystems), and nucleotide sequences were compared with belonging those to the prostanoid receptor family. A clone SN463 that showed significant receptor was selected as a cansequence homology with human didate, and its230-bp DNA insert was used as a probe for screeningby cross-hybridization. cDNA Cloning of Bovine PGF,,Receptor-cDNA was synthesized Fraction number from bovine corpus luteal poly(A)+RNA by Superscript and was fractionated by sucrosedensitygradientcentrifugation to separate the large DNA fragment. ThecDNA fraction, in which 5- to 6-kilobase pair DNA fragments were accumulated, was linked to EcoRI adaptor and subjected to construction of the cDNA library in A-ZAP11 vector. The phage blots on the nitrocellulose filter BAS85 were hybridized with "P-labeled SN463 230-bp DNA probe under stringent conditions.Fifteen singleclones were positively selected from 5.6 x lo5phage library by standard plaque hybridization. pBluescript plasmid DNA was excised from phage genome by coinfection of the helper phageR408, and 3 clones, BC2211, BC3111, and BC4111, carrying the largeDNA insert, were subjected to sequence analysis. Expression Assay of PGF,, Receptor in Xenopus Oocytes-Bovine corpus luteumpoly(A)*RNA was size-fractionatedby sucrose density gradient centrifugation (625%). A clone BC2211 digested by NotI or Sal1 was used as template DNA for in vitro transcription by T7 or T3 polymerase, respectively. Fractionated poly(A)+ RNA (50 ng) or in vitro T 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 synthesized RNA (1.74.3 ng) was injected intofollicle-enclosed Xenopus oocytes, and electrophysiological assay by measuring Ca2+-dependent inward C1- currents was done under a conventional 2-microelectrode voltage clamp a t -60 mV as described previously (26). Oocytes were perfused by a constant streamof perfusion buffer (90m~ NaCI, 1 mM KCl, 1 m~ CaCl,, 1 m~ MgC12, and 5 m HEPES pH 7.4). and PG dissolved in perfusion buffer was addedby switching the flow. PGF,, Receptor Expression in COS-7 Cells-The 2.2-kilobase pair -28s EcoRI DNA insert of BC2211 was subcloned into the NotI sites of pEFBOS eukaryotic expression vector (27) via the EcoRI-Not1 adaptor. To express the receptor protein transiently, we transfected the resultant plasmid DNA into COS-7 cells by the DEAE-dextranmethod (28). PGF,, binding assay was carried out essentially as described previously (29). COS-7 cells were plateda t approximately 1 x lo6 celldl5-cm plate and used for transfection. At 48 h aRer transfection,COS-7 cells were collected and membranes were prepared a s described (26). The membrane ( 2 5 4 0 pg) was incubated with5 ~IMb3H1PGF2, in theabsence or Fr. 16 Fr. 18 presence of various concentrations of unlabeled PG in100 pl of 10 m~ KP,, pH 6.0, and 100 m NaCl a t 23 "C for 1h. Formation of [3Hlinositol phosphate (InsP) was measured as described previously (30). Briefly, COS-7 cells seeded in6-well plates (5 x IO4 celldwell) were transfected Fr. 21 and grown for 24 h. After labeling the cells for another 24 h with F2. [3Hlinositol (2 pCi/well), the cells were washed twice with HEPESbuffered saline solution containing 125 m NaCl, 4.7 m~ KCI, 2.2 m~ CaCI,, 1.2 m~ MgCI,, 1.2 m~ KH,PO,, 15 m~ NaHC03, 11m glucose, and 15 m~ HEPES pH 7.4 and preincubated with HEPES-buffered saline solution supplemented with 10 m~ LiCl for 5 min at 37 "C. The reaction was started by the addition of various concentrationsof PGF,,. FIG. 1. Northern blot analysis and electrophyeiological aeeays After incubation for 10 min, the medium was quickly aspirated, and 2 ml of5% (w/v) trichloroacetic acid solution was added to each well. in Xenopus oocytes. Bovine corpus luteal poly(A)+ RNA was sizefractionated by 5-25% sucrose density gradient centrifugation.a, agaTrichloroacetic acid was removed from the extract twice with diethyl rose gel pattern of fractionated RNA. Each RNA fraction (0.9 pg/lane) ether and then the extract was neutralized with 150 pl of 0.2 M Tris. was separatedon 1% agarose gel and stained with ethidium bromide. b, Separation of [3HlInsPs was carried outby Bio-Rad AG 1 x 8 chroma- Northern blot hybridization. RNA blot on a NYTRAN membrane filter tography. was hybridized with 32P-labeled SN463 230-bp probe DNA. Fraction Northern Blot Analysis-Total RNAs were prepared from various numbers are shown on the top of the gel and blot. T represents total bovine tissues by the standard acid guanidinium thiocyanate method poly(A)+RNA of bovine corpus luteum. The positionsof 18 S and 28 S (31),and poly(A)* RNAs were purified by an Amersham's mRNA puri- rRNAs are indicated on the right. c, electrophysiological responses in fication kit. Poly(A)+RNAs (5 pg of each) from each tissue were elec- Xenopus oocytes by PGF2,. Fractionated RNAs (approximately 50 ng) trophoresed on 1.2% agarose gel and transferred onto a NYTRAN nylon were injected into oocytes, and responses to 10 p~ PGF,, were measmembrane filter. The230-bp DNA generated from SN463 by PCR was ured after 4 2 4 6 h. Current traces shown are recordingsFrom oocytes labeled by 32Pand used as a probe. Hybridization was carried outin a injected with fractions 16, 18, 20, and 21.

T X A ,

3883

Cloning of the Bovine PGF,, Receptor zolium,5-bromo-4-chloro-3-indolylphosphate and lavamisole, an inhibitor of endogenous alkaline phosphatase.

RESULTS AND DISCUSSION The combination of PCR screening using degenerated primersandsequential cross-hybridization gaveone full-length cDNA clone(BC2211) and two partial clones(BC3111 and BC4111)of receptor for PGF2- from a bovine corpus luteal cDNA library. Original designing of the degenerated primers for PCR was based on the commonly appeared motifs on the putative seven transmembrane domains of known prostanoid receptor family. Among the seven transmembrane domains (TDs), the nucleotide sequence homology was comparably well observed in the regions of TDIII, TDIV,TDVI, and TDVII. Sense primer for TDIII was derived from a common motif of known prostanoid receptor family. Another common motif, which is located on the downstream region of TDIV, was selected as a site for the second sense primer DGPIV. Two types of antisense primers for TDVI and TDVII were independently designed from the complementary sequences of 'zxA2/EP3(type A) and EP,/EP2 (type B) and were designated as DGPVIA,

DGPVIB, DGPVIIA, and DGPVIIB. BecausePGF2, receptor protein is highly expressed and abundantly accumulated in corpus luteum (17-20), we used corpus luteal mRNA as a library source. Template cDNAs were prepared from poly(A)+RNAs of bovine corpus luteum and also from nonchromaffincells of adrenal medulla (32) and heart as negative controls, by oligo(dT)-primedreverse transcriptase reaction. The annealing temperature for each PCRcycle was reduced to 48 "C to help the efficient annealing of degenerated primers. To find the bovine corpus luteum-specific DNA fragment, we compared the Southern gel blots of each PCR product after the sequential hybridization with probe DNAs of bovine T X A 2 and EP3 and mouse EP1 receptors (data not shown). Probe DNAsof TXA2 and EP3 receptor gave corpus luteumspecific 230-bp band (SN463), which was amplified with DGPIV and DGPVIB primers. The sequence analysis revealed that this SN463 fragment has 55% nucleotide sequence homology with a corresponding region of bovine TXA2 receptor cDNA. The deduced amino acid sequence also shared 36% homology with bovine T X A 2 receptor protein, especially higher homology CCGGCAGCTCTTATCTCCATMCA

-1

ATG TCC ACG AAC AGT TCT ATA CAG CCA GTG TCT CCT G M TCT GAG CTC CTT TCAM T ACA ACT TGC CAA CTG G M Met Ser Thr Asn Ser Ser Ile Gln Pro Val Ser Pro Glu Ser Glu Leu Leu Ser Asn Thr Thr Cys Gln Leu Glu

75 25

G I U GAC CTT TCAATA TCT TTT TCA ATA ATC TTC ATG ACA GTG GGA ATC TTA TCG M C AGC GCC ATT GCT Glu Asp Leu Ser Ile Ser Phe Ser Ile Ile Phe Met Thr Val Gly Ile Leu Ser Asn Ser Leu Ala Ile Ala Ile

150

TTT AGA CAG M G TAT AAG TCA TCG TTT TTG CTT TTG GCT AGT GCT CTA ATC GTA Phe Arg Gln Lys Tyr Lys Ser Ser Phe Leu Leu Leu Ala Ser Ala Leu Val Ile

225 75

t

CTC ATG AAG GCA TAC CAG AGA Leu Met Lys Ala Tyr Gln Arg II ACA GAT TTC TTTGGG CAC CTC Thr Asp Phe Phe Gly His Leu

I

t

50

ATC TAC Ile Tyr

300 100

TTT CTA Phe Leu

375 125

CAC TCT ACA AAA ATT ACA ACC M G His Ser Thr Lys Ile Thr Thr LYS

4 50 150

TTG CTG CCC ATC CTC GGG CAC CGT Leu Leu Pro Ile Leu Gly His Arg

525 175

GAC TAT M A ATT C M GCT TCA AGG ACC TGG TGT TTC TAC A M ACA GAT GAAATC A M GAC TGG G M GAT AGG TTT ASD Glu Ile Lvs ASD TKD Glu ASD Ara Asp Tyr Lys Ile Thr TKD Cvs Phe Tvr

200

TAT CTT TTA CTT TTT GCT TTTCTG GGG CTC CTA GCC CTG GGT ATT TCA TTT GTT TGC AAT GCC ATC ACA GGA ATT Tvr Leu Leu Leu Phe Ala Phe Leu Glv Ala Leu Glv Ile Ser Phe Val Cvs Asn Ala Ilc Thr Glv Ile

675 225

TCA CTT TTG AAA GTT AAA,TTTAGA AGT CAG CAG CAC AGA Ser Leu Leu Lvs Val Lvs Phe Ara Ser Gln Gln His Ara VI CTT CTG GGT ATA ATG TGT GTT TCC TGC ATT TGG TGT AGT Leu Leu Gly Ile Met C p Val Ser Cys Ile Cys Trp Ser

C M GGC AGG TCT CAT CAC TTT GAA ATG GTC ATC CAG Gln Glv Ara Ser HisHis Phe Glu Met Val Ile Gln

750 250

CCT TTT CTG GTG ACA ATG GCC AGC ATT GGA ATG AAT Pro Phe Leu Val Thr Met Ala Ser Ile Gly Met Asn

825 275

ATC M T GGA ACT ATA GCA GTC TTT GTG TAT GCT TCT GAT AAA GAC TGG Ile Asn Gly Thr Ile Ala Val Phe Val Tyr Ala Ser Asp Lys Asp Trp 111 TTT GAC AAG TCA AAT ATC CTT TGC AGT ATT TTT GGT ATC TGC ATG GTG TTC TCT GGT CTG TGC CCA CTT Phe Asp Lys Ser Asn Ile Leu Cys Ser Ile Phe Gly Ile Cys Met Val Phe Ser Gly Leu Cys Pro Leu GGC AGT TTG ATG GCC ATA GAG CGA TGC ATT GGA GTC ACC A M CCA ATA TTT Gly Ser Leu Met Ala Ile Glu Arg Cys Ile Gly Val Thr Lys Pro Ile Phe IV CAT GTT M A ATG ATG TTG AGT GGG GTG TGC TTTTTT GCT GTT TTT GTA GCf His Val Lys Met Met Leu Ser Gly Val Cys Phe Phe Ala Val Phe Val Ala

*

*

VI1

600

ATA CAA GAT TTTAAG GAT TCC TGT GAA AGA ACC CTT TTT ACT CTT CGA ATG GCA ACA TGG M T CAA ATC TTA GAT Ile Gln Asp Phe Lys Asp Ser Cys Glu Arg Thr Leu Phe Thr Leu Arg Met Ala Thr Trp Asn Gln Ile Leu Asp

900 300

CCT TGG GTG TAT ATT CTT CTA CGG AAG GCT GTC CTT AGG AAC CTA TAT GTG TGT ACC AGA CGC TGT TGTGGA GTA Pro Trp Val Tyr Ile Leu Leu ArgLys Ala Val Leu Arg Asn LeuTyr Val Cys Thr Arg Arg Cys Cys Gly Val

975 325

CAT GTC ATC AGC TTA CAT GTT TGG GAG CTT AGC TCC ATT A M GAT TCC TTA M G GTT GCT GCT ATT TCT GAT TTA His Val Ile Ser Leu His Val Trp Glu Leu Ser Ser Ile Lys Asp Ser Leu Lys Val Ala Ala Ile Ser Asp Leu

1050 350

*

CCA GTT ACA GAG A M GTA ACT CAG CAA ACA AGC ACC TAGTTGAATAGGATGGTAAATCAGTGTAGGTCTAGGACCAAA~AAAAAAA 1138 Pro Val Lys Thr Glu Val Thr Gln Gln Thr Ser Thr 362 AGTATTGGCAATACTTCAGTTAATTGTGTAAATAGAGAAAGTCTMCTGGAAAMTCAGCCCTCACCCAGATAGTATGGGGGCAC~TGTCAGACTTGG 1238 CTTTTAAATTTGTTAAATTAGCTGTTTAATTAATTACGTATTTTAACACGCTTTTGTCMCTGGAGGTAMTGTGAT~TMTGCCATAGGAGTCAAACGM 1338 AGCAGTTTGGGCCTTATCTGTGCTATTGTT~TTT~AAATGAG~ACTCTCTTGMGCC~MGCGTGTAT~GGTCTACTACCTGMCAGTGMATGG 1438 CCATTTAGGGGGTCACTGCTCTATGCAGACCMGCACAGTGAAA~GGGCACCTTCTTTGA~TGGTC~TTTTACTCCTCGTTACATAGCCTCAGTTAA 1538 TACGTGCGTTGTCATGTCACAGGGGATTGATTGTGGCTTCTTATM~GACTTCCACATACGTGGTTGGCTGTCAGGTTGCCTGGTCTTGTGAGCCTGTTT 1638 AGAATGAACGTTTCTTTGTCATATTT~ACAGATACMCTCTTGCATTTA~GATATGMGGTCAGTTTTTGTTTGMGATATTCTTGTTAAGTCATAGAT 1738 TTCCACAGTTTTCAAGTMTCACCATTTCCTCTG~TGCTTATGTATAGTCMTAGTTTGTAGAG~CAMGTCTGTGTGAGCAGGTGTAGAGGTGA 1838 CTTGCACTGTAGCGGTTCCTMGGMCTCAGCCATGGATMTGCAAACMGCAGMGTTAAACCCTMGTGATGGGTGCAGAGAACATTGGTAAAGGTGC 1938 TTTACCTGAGMCCATTGACACAGTGTCAmCTGTGTGTCAGGGMCAAAAGMACACAGCCTCTAGAG~ATATTCAAAGACCATCTGCAGCTAGTGT 2038 GTTTCMTTCACTTATACACGCACATATACTCACACACACACCMGGACATCAGAAATTTMGTTGMAGGAATTCTTTAAATCTGTMGATGGCATC~ 2138 CCMAGCCTGTACTACCMTGTCMGGGGM~T~GGCMTTAGCCAGGTATTTTGCCGG 2198

FIG.2. The nucleotide sequencefor the bovinePGFp, receptor (BC2211)and ita deduced amino acid sequence.The deduced amino acid sequence is shown below the nucleotide sequence. The ATG found in the most 5' end in the frame was assigned as an initiation site for translation. The bold underline indicates the locus of 230-bp DNAinsert of clone SN463. Positions of the seven putative transmembrane segments I-VI1 are tentatively assigned by hydropathy analysis andareindicatedabove the nucleotide sequence. The arrows indicate the potential N-glycosylation sites in the amino-terminal region;*, potential phosphorylation sites by protein kinase C.

Cloning Bovine of the

3884

PGF,, Receptor

40,983. According to the Kyte and Doolittle method (331, the being observed in theTDV locus. Inourpreliminaryexperiments, electrophysiological re- hydropathy profile of the deduced amino acid sequence was sponses to PGFz, stimulation in Xenopus oocytes were ob- analyzed, and itrevealed the seven hydrophobic segments that served in RNA fractions larger than 28 S rRNA, suggesting could represent putative transmembrane domains (data not that themRNA size for the bovine PGFz, receptor is as large as shown). The NH2-terminalregion of this receptor protein con5 kb. Therefore, poly(A)+ RNAof bovine corpus luteum was tains two potential N-glycosylatiap sites, as observed with size-fractionated by sucrose density gradient centrifugation for other members of G protein-coupled receptors (34). On the Northern blot hybridization (Fig. la). Fig. l b clearly indicated other hand, there are4 serine or threonine residues found in that SN463 DNA strongly hybridized to 5-kb mRNA in RNA the COOH-terminal end and 2 of them in the second intracelfractions 17-20. This hybridization pattern is well correlated lular locus, which could be potentially subjected to phosphorywith electrophysiological responses to PGF2, in Xenopus oo- lation by protein kinase C. The transmembrane segment VI1 residue, as commonly conserved in cytes injectedwith RNA fractions (Fig. IC), demonstrating that contains Arg-291 at the 4th SN463 mayencode a part of the bovine PGF2, receptor. There- the prostanoid receptor family (21-25). The sense RNA corresponding to the coding receptor protein fore, this SN463 DNA was selected as the best source of probe DNA for further screeningof the full-length clone. By the standard plaquehybridization, one full-length cDNA 1 2 3 4 5 6 7 8 9 10111213 clone, BC2211 and two partial clones, BC3111 and BC4111, were isolated. Fig. 2 shows the nucleotide sequence and deduced amino acid sequence derived from one representative clone BC2211. The open reading frame(1086 bp) consists of 362 amino acid residues with an estimated molecular weight of

-28s

I

1 rnin

FIG.3. Current traces recordedfrom Xenopus oocytes injected with in vitro synthesized mRNA. Cloned BC2211 was digested by Sal1 (antisense)or Not1 (sense) for the templateof in vitro transcription by T3 or T7 RNA polymerase, respectively. Cloned BC4111 was digested by Sal1 (sense) for the template by T3 polymerase. The synthesized mRNA was injected into a n oocyte, and response to 10 p~ PGF2, was examined after22-27 h. Electrophysiological measurement was done as described under "Experimental Procedures." The type and amount of mRNA injected into an oocyte are as follows: a, antisense mRNA of BC2211, 3.5 ng; 6, sense mRNA of BC4111, 4.3 ng; c, sense mRNA of BC2211, 1.7 ng.

18s

+

FIG.5. Northern blot analysisof poly(A)*RNA isolated from various bovine tissues and nonchromaffin cells. Experimental detailsare describedunder"Experimental Procedures." The poly(A)+ RNAs used (7 pg of each) were isolatedfrom the following tissues and cells: lune 1, liver; lune 2, ovary without corpus luteum;lune 3, uterus; lane 4, spleen; lune 5, large intestine; lane 6 , small intestine; lane 7, lung; lane 8,aorta; lane 9, cerebrum; lune 10, cerebellum; lune 11, stomach; lune 12, corpus luteum; lune 13, nonchromaffin cells of adrenal medulla.

E

-lw[PGadded (M)]

-log[PGRa (M)]

FIG.4. a , displacement of specific ["HIPGF,, binding to membranesof COS-7 cells transfected with receptorcDNA Binding of t3HIPGF2, was determined in the presence of indicated concentrationsof unlabeled PGF,, (0).PGD, (0).PGE, (A), STA, (O),9cr,llP-PGF2 (A),and cicapmst (m). Experimental detailsare described under "ExperimentalProcedures." 6, stimulation of [3H]InsP formationby PGF,, in cDNA-transfected (0)and untransfected ( 0 )COS-7 cells. Indicated concentrationsof PGF,, were addedto the reaction mixture and incubated with cells in a 6-well plate for 10 mina t 37 "C. 13HlInsP formation was determined a s described under "Experimental Procedures." Formation of total [3HlInsPs (InsP, plus InsPz plus InsP3) is expressed as a percentageof that without PGF,,. Each point represents the meane S.E. of triplicate determinations.

Cloning of the Bovine PGF,, Receptor

3885

FIG.6. In situ hybridization of bovine ovary with digoxigenin-labeled riboprobe forPGFap receptor. Bright field photomicrographs showing in situ hybridization of corpus luteum ( C L ) with digoxigenin-labeled antisense (panels a and b ) and sense (panel c) RNA probes especially in thecytoplasm of granulosa luteal cells ( b ) , synthesized from clone BC2211. Highly specific hybridization signal is seen inthe CL (a), while it is absent inpanel c. Bars, 1 mm (a), 50 pm ( b and c).

I bFP hTP mTP bEP3 mEP3

"~""""_" MWPNGS MWPNGTSLG~CFRPV-N&.QEFSAI MKATRDHSAAPFCTRFNHSDPGIWAAEW&AP LPPEPSEDCGS """_ ~ m E H S m & - - S ~ D D c G s " " " " " " " " " "

&NSSIQ

" " " " " " " " " " "

I1

111 111

bFP hTP mTP bEP3 mEP3 IV

bFP hTP mTP bEP3 mEP3

V

..

VI

bFP hTP mTP bEP3 mEP3 bFP hTP mTP bEP3 mEP3

FIG.7. Alignment ofthe deduced amino acid sequences among bovine PGF,, and other prostanoid receptors. Deduced amino acid sequences of the bovine PGF,, ( b F P ) ,human and mouse TXA,(hTP and rnTP), and bovine and mouse EP, (bEP3 and rnEP3) receptors are compared with and aligned to achieve the optimal homology. The conserved amino acids are boxed, and the putative transmembrane segments are indicated by upper lines. was transcribed from Not1 digested-BC2211 DNA and injected into a Xenopus oocyte. The electrophysiological response by PGF2, was measured by the 2-micropipette voltage clamp method. As shown in Fig. 3c, the typical responseby PGF2, was observed in the senseRNA-injected oocyte but not in the antisense RNA-injected one (Fig. 3a). The clone BC4111 carrying a partial sequence of the coding region showed no response (Fig. 36), suggesting that the clone BC2211 encoded a functional receptor protein for PGF2,. The electrophysiological responses to 10 PGD2 and 101.1~STA2were similarly observed in sense

RNA-injected oocytes (data not shown). To determine accurately the affinities of the PGF2, receptor for PGs, we examined the ligand-binding properties of the receptor expressed transiently in monkey kidney COS-7 cells after transfection of the cloned cDNA. The EcoRI fragment of BC2211 was inserted into theeukaryotic vector pEF-BOS, and this plasmid was introduced into COS-7 cells. The binding of [3H]PGF2, to the membranes preparedfrom the cDNA-transfected cells wasmeasured at 23 "C. Specific binding of [3H]PGF2ato the membranewas saturable, and the Scatchard

Cloning of the Bovine PGFZaReceptor

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plot analysis indicated the presence of a single binding site with a Kd value of 12.4 n~ (data not shown). This value agrees well with those of bovine and ovine corpus luteum reported previously (17,201.No such sites were detected on membranes prepared from untransfected cells. Fig. 4u shows the result of displacement of [3HlPGFz, binding to membranes derived from cDNA-transfected cells by various PGs. The binding of [3HlPGFz, was inhibited by unlabeled PGs in the order of PGFZ, > PGDz > FGEz > STA,, a stable T X A Z agonist. This characteristic of binding specificity was in good agreement with the PGFza receptor reported previously with corpus luteum of various species (5, 17, 20). Cicaprost, a specific PGIz agonist (39, could not displace L3HIPGFz, binding to the membrane. While 9a,llp-PGFz completely displaced [3H]PGF2, binding to bovine corpus luteal membranes at one order higher concentration than PGF,, (data not shown), it partially inhibited the binding to thecDNA-transfected membrane. This characterization clearly demonstrates the selectivity of the expressed binding site to PGFz, and some cross-reactivity with PGD, and PGEz. The binding characterization described here shows the properties of PGFz, receptor, which is encoded by a single functional cDNA. We next examined whether PGFzu stimulates [3H]InsP formation in COS-7 cells transiently expressing the receptor. During a 10-min incubation, PGF,, could stimulate the formation of [3HlInsP1and [3HlInsPzbut not [3HlInsP3(data not shown). As shown in Fig. 4b,PGF,, increased total [3H]InsPformation at higher concentrations in cDNA-transfected cells but not in untransfected cells. As shown in Fig. 5, Northern blot analysis demonstrates that 5-kb mRNA is abundantly expressed and accumulated in the corpus luteum butnot in theovary devoidof corpus luteum and other organs examined. Next we analyzed bovine ovary tissue for PGF,, receptor mRNA by in situ hybridization with riboprobe (Fig. 6). Consistent with the RNA blot analysis, the great majority of granulosa luteal cells in the corpus luteum exhibited specific hybridization signals with the antisense riboprobe, while the ovary constituent cells other than thecorpus luteum remained unlabeled. It is well accepted that PGFzu is the endogenous luteolytic hormone to induce luteolysis by inhibiting progesterone production and that the site of PGFza is directly on the corpus luteum (6,36). The present results clearly demonstrate that mRNA for the PGFz, receptor is specifically expressed in luteal cells and support the idea that prostanoidinduced luteolysis is mediated by the PGFzu receptor. As shown in Fig. 7, the sequence comparison analysis revealed the significant sequence homologyof PGF2, receptor with other members of the prostanoid receptor family, espereceptor protein. They share 34 and 33% cially with the amino acid homology with human (21) and mouse (37) TXAz receptor, respectively. Similar homology is observed with bovine and mouse EP3 receptor (31 and 32%, respectively). A percentage homologyof PGFz, receptor is much less with mouse EP1 (21%) or EPz (18%) receptor. The bovinePGFz, receptor contains comparably short third intracellular andextracellular segments as observed with TXAz receptor protein, the extracellular segment of the PGFZureceptor being still 8 residues shorter than that of the TXA,receptor. Although the consequences of PGFz,-induced luteolysis are well known, the mechanisms that lead to luteolysis are virtually unknown. The successful cloning of functional cDNA for PGFzureceptor presented in thisstudy has provided a complete

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amino acid sequence and the proof on the distribution of this receptor in vivo.The availability of specific cDNA for the PGFzu receptor should help in understanding the molecular mechanism of receptor function and facilitate studies on the regulation of PGF,, receptor gene expression in corpus luteum. Acknowledgments-We thank Dr. Shuh Narumiya of Kyoto University for the kindgift of plasmid clone for the humanTXA,receptor. We are gratefulto Dr. Shigekazu Nagataof Osaka Bioscience Institute for providing pEF-BOS vector and to Dr. Kazushige Sugama of Pharmacology Institute of Bayer Yakuhin for providing bovine corpus luteal cDNA library and for helpful discussions. We also thank Dr. Ryotaro Yoshida for helpful adviceand critical reading of this manuscript. REFERENCES 1. Moncada, S., Flower, R. J., and Vane, J. R. (1985) in Goodman and Gilman's the Pharmacological Basis of Thempeutics (Gilman, A. G., Goodman, L. S., Rall, T. W., and Murad, F., eds) 7th Ed., pp. 660-673, Macmillan Publishing, New York 2. AnggM, E., and Bergstrom, S . (1963)Acta Physiol. Scand. 66,1-12 3. AnggSrd, E., and Samuelsson, B. (1963)Acta Physiol. Scand. S9, Suppl. 213, 170 4. Perkins, E. S . (1975)Adv. Ophthalmol. 29.2-21 5. Coleman, R. A,, Kennedy, I., Humphrey, P.P. A., Bunce, K , and Lumley, P. (1989) in Comprehensive Medical Chemistry (Hansch, C., Sammes, P. G., Taylor, J. B., and Emmett, J. C., eds) Vol. 3, pp. 643-714, Pregamon Press, Oxford 6. Horton, E. W., and Poyser, N. L. (1976) Physiol. Rev. 56, 59-51 7. Jimenez de Asua, L., Clingan, D., and Ruland, P. S . (1975) Proc. Natl. A d . Sei. U. S. A. 72, 2724-2728 8. Hesketh, T. R., Moore, J. P., Moms, J. D. H., Taylor, M. V., Rogers, J., Smith, G . A,, and Metcalfe, J. C. (19851Nature 313,481484 9. Minami, T., Uda, R., Horipuchi, . S., Ito, S., Hyodo, M., and Havaishi, 0.(1992) Pain 60,223-229 10. Davis, J. S . , Weakland, L. L., Weiland, D. A,, Farese, R. V, and West, L. A. (1987)Proc. Natl. Acad. Sci. U. S.A. 84.37283732 11. Moenner, M., Magnaldo, I., L'Allemain, G., Barritault, D., and Pouysdgur, J. (1987) Biochem. Bioohvs. Res. Commun. 1 4 6 . 3 2 4 0 12. Hakeda, Y., Hotta, T.,*K;rihara, N., Ikeda, E:, Maeda, N., Y a w , Y., and Kumegawa, M. (1987)Endocrinology 121,1966-1974 13. Fukami, K., and Takenawa, T. (1989) J. Biol. Chem. 264,14985-14989 14. Ito, S.,Negishi, M., Sugama, K., Okuda-Ashitaka, E., and Hayaishi, 0.(1990) M u . Prostaglandin ThromboxaneLeukotriene Res. 21,371474 15. Kitanaka, J., Onoe. H., and Baba, A. (1991)Biochem. Biophys. Res. Commun. 178,946-952 16. Ito, S., Sugama, K., Inagaki, N., Fukui, H., Giles. H., Wada, H.,and Hayaishi, 0. (199% Glia 6,67174 17. Powell, W. S., Hammarstrom, S., and Samuelsson, B. (1975)Eur. J. Biochem. 56-73-77 ~18. Powell, W. S., Hammarstrtim, S., and Samuelsson, B. (1976) Eur: J. Biochem. 61,605-611 19. Kyldh, U., and Hammarstriim, S . (1980) Eur: J. Biochem. 109,489-494 20. Balapure, A. K , Rexroad, C. E., Kawada, K., Watt, D. S., and Fitz, T. A. (1989) Biochem. Phnrmacol. 38,23752381 21. Hirata, M., Hayashi, Y., Ushikubi, F., Yokota, Y.,Kageyama, R., Nakaniahi, S., and Narumiya, S . (1991) Nature 349,617420 22. Sugimoto,Y., Namba, T., Honda, A,, Hayashi,Y., Negishi, M., 1chikawa.A.. and Narumiya, S . (1992) J. Biol. Chem. 287, 6463-6466 23. Honda, A., Sugimoto, Y., Namba, T., Watabe, A,, Irie, A,, Negishi, M., Narumiya, S., and Ichikawa, A. (1993) J. Biol. Chem. 288,7759-7762 24. Watabe, A,, Sugimoto, Y., Honda, A,, Irie, A., Namba, T.,Negishi, M., Ito, S., Narumiya, S., and Ichikawa, A. (1993)J. Biol. Chem. 268,20175-20178 25. Namba, T.,Sugimoto,Y., Negishi, M.,Irie, A,, Ushikubi, F., Ito, S., Ichikawa, A,, and Narumiya, S . (1993) Nature 365, 166-170 26. Yamashita, M., Fukui, H., Sugama, K, Horio, Y., Ito, S . , Mizuguchi, H., and Wada, H. (1991)Proc.Natl. Acad. Sci. U. S.A. 88, 11515-11519 27. Mizushima, S., and Nagata, S . (1990)Nucleic Acids Res. 18,5322 28. Cullen, B.R. (1987) Methods Ewymol. lS2,684-704 29. Negishi, M., Ito, S., Tanaka, T., Yokohama, H.,Hayashi, H., Katada, T.. Ui, M., and Hayaishi, 0.(1987) J. Biol. Chem. 262, 12077-12064 30. Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, 0. (1988)J. Biol. Chem. 263,111!3-1122 31. Chomczynski, P., and Sacchi, N. (1987) A n a l . Biochem. 162,156-160 32. Okuda-Ashitaka, E.,Sakamoto, K, Giles, H., Ito, S., and Hayaishi. 0.(1993) Biochim. Biophys. Acta 1176,148-154 33. Kyte, J., and Doolittle, R.F. (1982)J. Mol. Biol. 167, 105132 34. Hubbard, S . C., and Ivatt, R. J. (1981)Annu. Rev. Biochem. 6 0 , 5 5 5 3 8 3 35. Lawrence, R., Jones, R., and Wilson, N. (1992)Br:J . Phanarol. 106,271-278 36. Dorfiinger, L. J., Luborsky, J. L., Gore, S . D., and Behrman, H. R. (1983) Mol. Cell. Endoerinol. 39, 225-241 37. Namba, T., Sugimoto, Y., Hirata, M., Hayashi, Y., Honda, A., Watabe, A, Negishi, M., Ichikawa, A., and Narumiya, S. (1992)B k h e m . Biophys. Res. Commun. 184, 1197-1203 ~~I

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