Alternative Splicing Generates Multiple Isoforms of a Rabbit ...

Vol. 269,No. 8, Issue of Februaly 25,pp. 6163-6169,1994 Printed in U.S.A.

THEJ O U ~ A OFLB I O ~ I CCHEMISTRY AL

Alternative Splicing Generates Multiple Isoforms of a Rabbit Prostaglandin E2Receptor* (Received forpublication, July 21, 1993, and in revised form, November 8, 1993)

Richard M. BreyerS$&Ronald B. Emesong, Jia-Liang Tarn&$, Matthew D. BreyerSII**, Linda S . Davis$, Richard M. AbromsonS, and SuzanneM. FerrenbachS From the mivision of Nephrology, the $Department of Pharmacology, the IIVeterans Administration Medical Center, and the Vanderbilt University School of Medicine, Nashville, lknnessee 37232-2372

hemodynamics (6)as well as water and ion transport in the thick ascending limb (7) and collecting duct. In the cortical collecting duct, PGE2 exhibits three major functional transport effects as follows: inhibition of sodium transport viaits ability to elevate intracellular Ca2+ concentration(€9, stimulation of waterreabsorption via increasing the intracellular level of CAMP (9, lo), and suppression of arginine-vasopressin water reabsorption by inhibiting CAMP generation (9).Accumulating evidence suggests that PGE2 exertsits cellular effects through specific receptors. Based upon the activity profile of PGE2 analogs in smooth muscle, four subtypes of PGE2 receptors, EP1, EP2, EP3, and EP4, have been proposed (1, 3, 11-13). EP1 causes smooth muscle contraction, while EP2 and EP4 cause smooth muscle relaxation. The EP3 receptor can cause either smooth muscle contraction or relaxation depending upon the tissue type and the experimental setting (3,141. Recently, it has been suggestedthat an EP3receptor mediates PGE2 inhibition of water absorption in thecortical collecting duct (15). Some PGE2 receptors have been shown to couple to their intracellular effectors via guanine nucleotide regulatory proteins (G-proteins) (16-181, and a cloned cDNA encoding a murine PGE2 receptor has been demonstrated to couple to Gi when transfected in vitro (19).This receptor, which was cloned from the P815 mastocytoma cell line, has a pharmacological profile consistent with thatof an EP3receptor subtype. A second isodescribed (20).The diversity form of this receptor has also been of physiologic effects demonstrated by EP3-selective ligands may be the result of a single receptor coupled to multiple signaling pathwaysor to multiplereceptors with similar pharmaProstaglandin E2 (PGE2)’ is a potent modulator of a wide cological profiles. To investigate themolecular mechanism of PGE2 actions,we variety of physiological responses, including inflammation (11, gastric acid secretion (2), vascular tone (31, lipolysis (4), and have cloned the renal PGE2 receptors. We describe here the a well molecular cloning of four E P receptors from a rabbit kidney water andion transport (5).The rabbit kidney represents characterized physiological model with respect to PGE2 effects. cortex cDNA library. The primary structure and ligand binding existence of a In the kidney, PGE2 has been shown to modulate glomerular profile of these receptors are consistent with the family of renal EP3receptors, which differ exclusively in their * Support for this project was provided in part by National Institutes COOH-terminal regions. The ligand binding profile and tissue distribution of these receptors is consistent with a functional of Health Grants DK-46205-01(to R. M. B.) and DK-39261-05(to M. D. B.), an American Cancer SocietyInstitutional Research Grant IN-25-33 role for this family of EP3 receptors in mediating the renal (to R. M. B.), and a Veterans Administration MeritAward (to M. D. B.). The costs of publication of this article were defrayed in part by the actions of prostaglandins aswell as the effects of prostaglandpayment of page charges. This article must thereforebe hereby marked ins on gastric acid secretion and adrenal function. “advertisement” in accordancewith 18 U.S.C.Section1734solely to indicate this fact. EXPERIMENTALPROCEDURES The nucleotide sequence(sJ reported in this paperhas been submitted Materials” & B 28767, butaprost, sulprostone, and 8-epi-PGF,, to the GenBank-IEMBL Data Bank with accession number(s) U04273, were generous gifts of Drs. M. P. L. Caton of Rhone-Poulenc Ltd., P. J. U04274, U04275, U04276. ll To whom correspondence should be addressed. Tel.: 615-343-8496; Gardiner of Bayer U. K., Dr. Rubanie of Berlex Laboratories,and Dr. J. Morrow, Department of Pharmacology, Vanderbilt University, respecFax: 615-343-7156. ** Recipient of a Department of Veterans Mairs Career Development tively. L3HlPGE, was purchased from DuPont NEN. Unlabeled PGE2, PGE,, PGF2., and PGD, were purchased from Cayman Chemical (Ann Award. The abbreviationsused are: PG, prostaglandin;G-proteins, guanine Arbor, MI). RT-PCR-Total RNA was isolated from freshly dissected kidney cornucleotide regulatory proteins; RT-PCR,reverse transcription polymerase chain reaction; kb, kilobase(s); nt, nucleotidek);UTR, untranslated tex or medulla from female New Zealand White rabbits using a modiregion. fication of the method of Chirgwin et al. (21). Poly(A)+RNA was pre-

Four cDNA clones homologouswith a murine prostaglandin E2 receptor have been isolated from a rabbit kidney cortex cDNA library. These cDNAs encode related proteins that differ only in their COOH-terminal sequences. Southern blot analysis of rabbit genomic DNA indicates that these receptor cDNAs represent alternatively spliced variants derived from a single gene. This was confirmedby isolation and sequence analysis of genomic clonescontaining common region exonsand unique 3’-coding exons, which contained introdexon boundaries at the predicted splice junctions. Transient expression of a novel full-lengthcDNAin COS1 cells confirmed the ligand binding profile typical of an EP3 receptor subtype. Ribonucleaseprotection assays indicate that the gene encoding these receptors is most highly expressed in kidney, adrenal, and stomach with lower but significant expression in uterus, lung, heart, ileum, spleen, and brain. Moreover, each of the cloned isoforms is expressed in thekidney. In situ hybridization analyses of rabbit kidney demonstrated that thelevel of expression is highest in the outermedulla with lesser expression in the cortex and no detectable expression in the inner medulla. Theligand binding profile and tissue distribution of these receptors is consistent with a functional role for this family of EP3 receptors in mediating the renalactions of prostaglandins as well as the effects of prostaglandins on gastric acid secretion and adrenal function.

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pared by two rounds of affinity purification on oligo(dT) cellulose spin fragments were separated on a 4% polyacrylamide, 7 M urea gel, folcolumns (Pharmacia LKB Biotechnology Inc.) using no-salt buffer to lowed by autoradiography. elute the poly(A)' RNA according t o the manufacturer's instructions. In Situ Hybridization-Zn situ hybridization was performed by the Reverse transcriptionPCR was carried out essentially as described (22), method of Pelton et al. (26). Rabbit kidneys were perfused, fixed in using 250 ng of poly(A)' RNAa s a template and oligonucleotide primers paraformaldehyde, and embedded in paraffin. 7-pm sections were cut derived from the mouse EP3a sequence (19). The sequences of the and hybridized at 50-55 "C for -18 h (see Ref. 26). Following hybridoligonucleotide pairs 5' TGC TGG GCG TGG GCC GCT ACA 3' and 5' ization, sections were washedat 50 "C in 50% formamide, 2x SSC, 100 ACC CAG GGA TCC AAG ATC TGG 3' and5' GAC AGTGTT CGG GCT m 6-mercaptoethanol for 60 min, treated with RNase A (10 pg/ml, 37 "C, 30 min), and washed in 10 mM Tris, 5 mM EDTA, 500 nm NaCl ATC CTC 3' and 5' GAC CAA CAG ACG GAG AGC ACA 3' were usedt o amplify products E P 506-963 and E P 342-816 (using the mouse EP3a (37 "C),2 x SSC (50 "C), and 0.1x SSC (50 "C). Slides were dehydrated m ammonium acetate. Photomicrographs numbering system), respectively. These fragments, representing over- with ethanol containing 300 lapping regions of transmembrane domains 111-VII, were cloned into were taken from slidesdipped in emulsion(Ilford K5, Knutsford, Cheshire,United Kingdom) diluted 1:l with 2% glycerouwaterand plasmid pCRII (Invitrogen). exposed for 7 days at 4 "C. After development in Kodak D-19, slides Full-length coding regions for clones 74A and 80A were amplifiedby RT-PCR using poly(A)' RNA isolated from renal medulla as template. were counterstained with hematoxylin and eosin. Photomicrographs were taken with a Zeiss Axioskop microscope using dark field optics. The upstream oligonucleotide primer was derived from the 5'-UTR common to clones72A and 77A (5' ACC ACG CCG CGT CTG CC A 3'), and the downstream primers were derived from the unique 3'-UTRs of RESULTS clones 74A (5' TTT TTC TCC ACT GCT CTC 3') and80A (5' GAG AGT Cloning of a Renal EP Receptor-A rabbit renalcortex cDNA ATA AGG GTT CAA 3'1, respectively. cDNA Cloning-A rabbit renal cortexcDNA library (AZAPII; Strata- library was screened with PCR fragments E P 506-963 and EP gene) was screened with the PCR fragmentsEP 506-963 and E P 342- 342-816 (using the mouse EP3a numbering system(19)). Five 816. lo6 clones were screened with probes labeledby random priming independent clones were isolated. The complete nucleotide se(23). Positive plaques were purified, and isolated cDNA-bearing phage quence of clone 72A is shown in Fig. lA. This clone contains a were rescued to the plasmid form using an M13 helper phage. Clones cDNA of 2061 nt and has an open reading frameof 361 codons were analyzedby restriction digestion and sequence analyses using the dideoxy chain termination method (24) on double-stranded template. encoding a full-length receptor with a n in-frame stop codon in Genomic Cloning-A rabbit genomic DNA library (ADash; Strata- the 5"untranslated region. There are two potential translaof the first transmembrane domain; gene) was screened with the full-length cDNA 72Aand PCR fragments tional start sites upstream generated from the unique3' sequences of cDNA clones 80A, 77A, and the initiator ATGof clone 72A schematized in Fig. 1 most 74A. 5 x lo5 clones were screened with probes labeled by random prim- closely matches the consensus sequence for translation initiaing (23). Positive plaques were purified and isolated and restriction tion (27). Analysis of the predicted amino acid sequence indifragmentscontaining exon-specific sequences subcloned intopBluescript SK-. Clones were sequencedas above using oligonucleotide prim- cates that the polypeptide encoded by this cDNA has seven transmembrane regions typical of G-protein-coupled receptors ers derived from cDNA sequences. COSl Dansfection-A full-length 2.1-kb cDNA fragment (clone 72A) and is82% homologous to themouse EP3a receptor (19). There was subcloned into the mammalian expression vector pcDNAIneo (In- is 92% sequence identity of the mouse and rabbit transmemvitrogen) and the resultant plasmid transfected into COSl cells using brane (ligand binding) domains, with somewhat lower homolthe lipofectin method (Life Technologies, Inc.), using 10 pg of plasmid ogy in the NHz-terminal and extracellular loop sequences. DNA and 40 pg of lipofectin. Cells were culturedfor 72 h, changing the In addition to the ubiquitous pair of potential N-linked glymedium every 24 h. Cells were scraped into buffer containing 15 nm cosylation sites found in the NHz-terminalsequence of G-proHEPES, 5 nm EGTA, 5 m EDTA, 40 p~ indomethacin, and 2 mM phenylmethylsulfonyl fluoride and lysed by passage througha 21-gauge tein-coupled receptors (28,29),there aretwo additional potenneedle. The lysate wasthen centrifuged a t 200,000 x g for 1 h and total tial N-linked glycosylation sites on the extracellular surface membranes collected on a 60% sucrose cushion. (30), one in each of the second and third extracellular loops Ligand Binding Studies-For saturation isotherm experiments, 40 (Fig. lA). The COOH-terminal sequence of clone 72A is considpg of membrane protein was incubated in Buffer A (25 nm KP04,pH erably shorter than that of murine EP3a or EP3P. This region 6.2, 10 m MgC12, and 1 nm EDTA) for 2 h at 30 "C with various concentrations of [3H]PGE2.Nonspecific binding was determined in theis identical to the mouse sequences proximal to Gln-355 and presence of 50 unlabeled PGE,. Reactions were stopped by addition has no homology distal to this residue. of 3 ml of ice-cold Buffer A followed by rapid filtration on Whatman A Family of Alternatively Spliced Receptor VariantsGF/F glass fiber filters. Filters were washed three times with Buffer A, Sequence analyses of the remainingcDNA clones indicate that dried, and counted in DuPont 989 fluor. For competition assays, 80 pg they fall into four classes. Each class is identical in the common of membrane protein was incubated with 1 nM C3H1PGE2and various region, 5' to nucleotide 1065 (encoding Gln-355) using theclone concentrations of unlabeled competitor and reactions carried out as 72A numbering system (Fig. lA).The four classes of receptors above. DNA Analysis by Southern Hybridization-Rabbit blood genomic clearly diverge in sequence 3' to nucleotide 1065. Clone 77A DNA was purified by chromatography using Qiagen columns. 10 pgof encodes a full-length receptor, which is nearly identical to 72A genomic DNA was digested with the indicated restriction enzyme and throughout the common region; however, it diverges in the size-fractionated on a 0.8% agarose gel. Following alkali denaturation, variable region after residueGln-355. Clone 77Ahas two single the DNA was transferred to Nytran membranes (Schleicher & Schuell), base changes in the common region relative to clone 72A, a vacuum dried, and probed with E P 342-816 at high stringency (0.1 x silent mutation a t Gly-193 and an Ala to Thr substitution at SSC, 0.1% SDSat 65 "C). Ribonuclease Protection-For generation of RNase probes, fragment position 243. These variations may represent sequence polyEP 506-963 (common region) or fragments containing both common and morphisms. At the nucleotide level, the variable region of clone unique 3' sequences (see Fig. 1B)were amplified by PCR and cloned 77A has an insertionof 170 base pairs in theCOOH-terminal into the transcription vector pCRII. Isoform-specific protection probes sequence relative to clone 72A (Fig. 1).Moreover, the untransprimer in the common region (5' were generated using an upstream lated sequences present in clone 77A are identical to a portion ACA TCA GTT GAG CAC TGC 3') and downstream primers derived of the uniqueregion of clone 72A, suggesting that the inserted from the unique 3' region of clones 72A (5' AAC CAT TTA ATG GAT sequence in clone 77A is the result of alternative splicing. GCA 3'), 77A (5' GAT AAG GAC GAG CAC CAG 3'), 74A (5' TTT TTC In addition to the clones 72A and 77A, which contain fullTCC ACT GCT CTC 3'1, and 80A (5' GGG TGT AAT AAG GAAATA 3'1, respectively. Ribonuclease protection assays were performed essentially length coding regions, two partial cDNA clones have been isoas described (25).Antisense RNA was transcribed from the flanking T7 lated. These clones, 74A and 80A, contain complete variable or SP6 promoters, depending upon the fragment orientation, in the presence of [a-3ZP]UTP.The antisense RNA (5 x lo5 cpm) was hybrid- region coding sequences but are missing a portion of the 5' ized to 10 pg of total cellularRNA at 45 "C for 14-18 h. Ribonuclease A common region (Fig. 1).Clone 74A shares 70% amino acid idendigestion (20 pg/ml) was carried outa t 30 "C for 45 min, and protected tity with themouse EP3a clone in thevariable region (19) and

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FIG.1. A , the nucleotide and predicted amino acid sequence of clones 72A, 74A, 77A, and 80A. The deduced amino acid sequence is shown below the nucleotide sequence using the single-letter code. Positions of the seven putative transmembranedomainsare underlined inthe amino acid sequence. The boundaries of thetransmembraneregionsaredeterminedbaseduponhydropathyanalysis and comparison with other seven transmembrane receptors. Asterisks mark potential N-linked glycosylation sites in the extracellular regions.Arrows indicate the 5' terminus of the partial cDNA coding regions for clones 74A and 80A. B , a schematic alignment of the four EP receptor clones representing the four classes of sequence. The approximate position of the seven transmembrane domains is shown above. The coding region of each clone is shown as thicker regions of the bars, and regions of similarshading indicateregions of identical sequences.Beneath each clone, a schematic representation of the PCR fragment used in the isoform-specific RNase protection experiments is shown. 72A appears to be a full-length cDNA, 77A has the entire coding region, and clones 74A and 80A are fragmentsencoding only a portion of the corresponding receptor. The missing 5' regions of clones 74A and 80A amplified by RT-PCR are shown in dottedoutline.Arrows indicatetheapproximate position of the PCR primers used to amplify the full-length coding regions.

B

-

P 72A

is a likely candidate for the rabbit homolog of that receptor. Importantly, the 3"untranslated region of clone 74A contains the exon that encodes the variable region amino acids of clone 77A (Fig. 1B).A 91-base pair insertion in clone 74A, as com-

pared with 77A, encodes the variable COOH-terminal amino acids of this isoform. Clone 80A has a variable COOH-terminal region, which does not share sequence similarity with 72A, 77A, 74A, or the murine EP3 a or p isoforms.

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Full-length coding regions of clones 74A and 80A were amplified from renal medulla using an RT-PCR strategy, which presumed that the 5'-UTR and coding region of 74A and 80A would be identical to that of full-length clones 72A and 77A.

-23.1 -9.4 -4.4 6.6

-2.3 2.0

-1.3 -1.o -0.8 FIG.2. Southern blot of 10 pg of rabbit genomicDNA digested with the indicated restriction enzymes. DNA was size fractionated on a 0.8% aearose eel. transferred to a nvlon membrane.Drobed with EP 342-816 asYdescribYdin "Experimental~Procedures." I '

Using a sense oligonucleotide primer derived from the 5'-UTR of clones 72N77A and antisense primers derived from the unique 3'-TJl'R of 74A and 80A, respectively, full-length coding regions for 74A and 80A were amplified (Fig. m).Sequence analyses of the resultantproducts indicate that themissing 5' region of clones 74A and 80A were encoded by exons common to clones 72A and 77A. The Structure of the EP Receptor Gene-Cknomic Southern blot analysis with probe E P 342-816, which lies within the common region of the four cDNA clones, hybridizes to a single DNA fragment in each restriction digest tested (Fig. 2). Since the minimumsize encoding a protein of 361 aminoacids (72A) PstI, and PuuII are particularly is 1.1kb, digestions withAuaI1, informative, becausethe hybridizing fragments, 1.3and 0.8 kb, are too small to contain multiple receptor common regions in tandem. This is consistentwith the existence of a single common region gene, which isalternatively spliced. Sequence analyses of rabbit genomic clones indicate that the common coding region of EP3 is comprised of two exons and that the probe E P 342-816 lies within a single exon. Moreover, a n introdexon boundary was found at nucleotide 1065, the position where the proposed alternative splice junction of the EP3 cDNAs occurs (Table I). One clone contained the exon encoding 1kb downstream of the uniquesequences of 80A approximately .. the second common region exon. Several additional clones, which were isolated, contained exons encoding the unique 3' sequences of CDNAs 72& 7 7 4 a n d a fragment of 74A. The genomic sequence immediately preceding the unique 3' exons

TABLE I EP3 splice junctions Sequences of the intron-exon boundariesfor the common and unique receptorexons are shown.Exon sequences are shownin uppercase letters, and intron sequences are shown in lowercase letters. donor

Common 80A acceptor 77A acceptor 72A acceptor

...TTTTGCCAGIgtagca . . .

...tcccaccaacaglCATTCTCCAGCC . . . . . .accctttctaagIGTAATTCATG AA... ... tgttttgttcagIGAGGAATTTTGG . . .

[Competitor] M FIG.3. kftpanel, displacement of 1 IIM PHIPGE, with the indicated concentrationof naturally occurring prostaglandins.0,PGE,; A, PGE,; 0, PGF,,; 0 ,PGD,; +, 8-epi-PGF2,.Right panel,displacement of 1 IIM [3H]PGE2with the indicated concentrationof subtype selective prostaglandin inhibitors. 0,M & B 28767; *, sulprostone; x, butaprost.

Multiple PGE, Receptor Isoforms

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closely match theconsensus splice acceptor sequences, consist- A ent with alternative 3’ splice site selection (31). Expression of Clone 72A in Mammalian Cell Culture-To evaluate the ligand binding properties of this family of receptors, one of the full-length clones, 72A, was expressed in vitro and characterized. Clone 72A was transfected intoCOS1 cells, and membranes were prepared from these cells as described under “Experimental Procedures.” Saturation isotherms with [3HlPGE2yielded a KD of 320 PM and a B,, of 250 fmoVmg of membrane protein. No [3H]PGE2 binding was observed in either untransfected cells or cells transfected with the vector alone (data notshown). Competition experiments witha panel -3% of natural and syntheticprostanoid analogs indicate that this ”3441 clone has an order of affinity as follows: M & B 28767 > sul298 prostone 2 PGEz = PGEl > PGF,, > PGD2 > 8-epi-PGF2, (Fig. 3). Clone 72A binds theEP3-selective ligandM & B 28767 with the highestaffinity, followed by the EPlW-selective ligand sulprostone. The receptor did not bind butaprost, a n EP2-selective agonist. These binding properties are ingood agreement with B PROBE 77A 72A 80A 74A in vivo functional agonist order of potency of the rabbit renal Gi-coupled EP3 receptor (9, 15, 32). Expression of the EP Receptors in Vivo-An antisense RNA transcript of PCR fragment E P 506-963 protected a fragment of the expected size (466 nt) ina number of tissues inribonuclease ” protection assays (Fig. 4A). The location of the probe (transmembranes IV-VII) is within the shared region of the receptors c and would be expected to protect RNA transcripts corresponding to all of the isolated cDNA clones. This receptor geneis most highly expressed in kidney, followed by adrenal and stomach, with lower but significant expression in uterus, lung, heart, ileum, spleen, and brain. = r RNase protection probesspecific for each of the receptor isoforms were amplified by PCR from the corresponding cDNA clones. Each probe contains 150 n t of common sequence contiguous to a portion of the unique COOH-terminal sequence from 165 to 279 n t in length (Fig. lB). These probeswere FIG.4. A, ribonuclease protection of total RNA isolated from various radiolabeled and used to protect RNA isolated from kidney tissues. Antisense RNA labeled with [d2P1UTP was transcribed from medulla. For each probe, a full-length protected fragment was probe EP 506-963 and the protection assay performed as described observed, indicating that the corresponding mRNA encoding under “Experimental Procedures.”RNA protected 10 pg of total RNA from each of the indicated tissues. Radiolabeled DNAmarker sizes (1-kb each isoform is normally expressed in vivo in the kidney (Fig. ladder; Life Technologies, Inc.) are indicated in nucleotides. B, RNase 4B). Smaller fragments were also protected by each of the protection using isofom-specific probes. Protection was carried out as probes. Probe 77A protects not only the full-length 314-nt frag- above, using 10 pg of kidney medulla total RNA and antisense probes ment but a 150-nt species, which corresponds to the expected transcribed from isofom-specific fragments as described in the text (see Fig. lB). size of the common region resulting from hybridization to other mRNA transcripts derived from this gene. Hybridization of probe 77A to mRNA encoding any of the otherclasses of clones the outer medulla, with selective labeling of tubules. Lower would result inonly the 150-nt common region being protected. expression levels werefound in thecortex, where labeling was Probe 72A protects three species, the 420-nt full-lengthspecies, limited to distal tubules,with no detectable labeling of proxithe 150-nt common fragment, and a 270-nt fragment of the mal tubules, glomeruli, or blood vessels. No detectable expresexpected size for the uniquesequence of 72Arepresented in this sion was observed in the innermedulla. probe. The latter two fragments would result from hybridizaDISCUSSION tion of probe 72A to transcripts encoding clone 77A, where both We have cloned a family of E P receptor cDNAs from rabbit common and unique fragmentsare protected separately as the probe sequences are noncontiguous in 77A mRNA(see Fig. lB1. kidney. Sequence analyses of these cDNA clones suggested that In contrast,probes 80Aand 74Agenerate several fragments not they represent alternativelyspliced variants of a common preaccounted for by the four isoforms reported here. Probe 8OA mRNA. The genomic Southern analysis provides direct eviprotects a full-length fragment of 409 nt, the 150-nt RNA spe- dence that themultiple EP3 receptor isoforms are derived from cies and an additional fragment (260 nt) of unknown origin. In a single gene. Isolation and sequence analyses of multiple addition to the expected full-length 431- and 150-nt fragments clones isolated from a rabbit genomic library have demonprotected by74A, probe 74A protects three additional frag- strated the existence of consensus splice sites consistent with ments of 250, 190, and 165 nt. These results,utilizing probes RNA processing events. Two of the cDNA clones, 74A and 80A, 74A and SOA, strongly suggest that there are additional splice contained incomplete coding regions; however, full-length codvariants generated in kidney medulla that have yet tobe iso- ing regions of these clones were amplified byRT-PCR from rabbit kidney medulla. The sequence identity of the 5‘-UTR lated. To examine the intrarenal distribution of EP3 receptor gene found in each of the four isoforms further supports the notion expression, we hybridized probe E P 506-963 to sections of rab- that they are derived from a single gene. RNase protection bit kidney (Fig. 5). Highest levels of expression were found in experiments confirm that all of the four cloned EP3 splice vari-

F

-

= .

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Multiple PGEz Receptor Isoforms

Flc. 5. Zn situ hybridization of ““S-labeledprobe EP 506-963 to rabbit kidney cortex (upper panel, C ) and inner (lower panel, ZM)and outer (lower panel, O M )medulla as described under “ExperimentalProcedures.”Shown are darkfield exposures a t 50 x magnification.

ants are expressed in kidney medulla and also suggest that other unique isoforms may be present. The alternative splicing of messenger RNA precursors represents a common mechanism for increasing the flexibility of eukaryotic gene expression by the generation of structurally distinct, differentially regulated isoforms. Recent studies have indicated that alternative splicing is responsible for the generation of a number of proteins, including the peptide neurotransmitters calcitonidCGRP (33), tachykinins(341, ion channels (351, and growth factors(36). Alternative splicing has been observed in several G-protein-coupled receptors, including the third cytoplasmic loop of the dopamine D2 receptor (37,38), the cytoplasmic tail of the metabotropic glutamate receptor (39), and the murine EP3receptors (40). The COOH-terminal tail region of G-protein-coupled receptors is implicated in the regulation of receptor desensitization

by a number of protein kinases (41-43). In this regard, it is interesting thatclones 77Aand 80Aeach have a unique protein kinase C phosphorylation consensus sequence (44), whereas 72A and 74A do not. Indeed, clone 72A is unique in that it has no serine, threonine,or tyrosine residues to serve as targets of COOH-terminal phosphorylation by any of the kinases. Previous studies have demonstrated that the mouse EP3a receptor undergoes ligand-mediated desensitization, whereas the EP3P form does not (40). It is possible that the COOH-terminal regions of the rabbit EP3 receptor may also play an analogous regulatory role to that described for the murine system. Currently, there isno evidence to suggest that EP3couples to signal transduction pathways other thandecreasing CAMPvia Gi in the rabbit cortical collecting duct (45). However, recent studies with cloned bovine EP3 isoforms demonstrate coupling to multiple G-proteins when expressedinChinese hamster ovary cells (46). Previous studies have demonstrated promiscuous couplingof receptors that couple exclusively to Gi in vivo when these receptors are overexpressed in vitro.For example, the Gi-coupled a2* adrenergic receptor can couple to G,- and IPS-mediated signal transduction pathwayswhen expressed in vitro (47, 48). This relaxed specificity for G-proteins has been observed for receptors overexpressed in Chinese hamster ovary cells, though not in other cell types (47). There is substantial evidence that receptor/G-protein interactions are considerably more stringent in vivo(49-51). Whether EP3 receptors couple to multiple signaling pathwaysin vivoremains an open question. The EP3 receptor gene is most highly expressed in the kidney, followedby adrenal andstomach. Important roles for PGEz in each of these tissues have been suggested. The Gi-coupled PGE2 receptor (EP3) has been characterized biochemically in bovine adrenal medulla (52, 53) where it modulates catecholamine release. Pharmacologic studies also suggest an important role for EP3 in mediating gastricacid secretion (2, 54). In thekidney, PGE2 potentlyregulates salt and water transport. Specific labeling of tubules was especially high in the outer medulla, which is comprised of thick ascending limb and collecting ducts. It is well established that PGE2 potently inhibits NaCl and water reabsorption in both of these nephron segments (10, 15).These results suggest an important role for EP3 receptors in regulating renal epithelial transport. In support of this hypothesis, it hasrecently been suggested that an EP3 receptor inhibits vasopressin-stimulated water absorption in the collecting duct (15).The significance of the existence of multiple EP3isoforms in this tissue remainsto be established. Acknowledgments-We thank H. Jacobson for support and encouragement throughout this work, P. Bennet and L. Limbird for helpful discussions, and A. George, J . Capdevila, and J. P. Breyer for critical reading of the manuscript. REFERENCES

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