Proc. Natl. Acad. Sci. USAVol. 91, pp. 9208-9212, September 1994Medical Sciences

Molecular cloning of a potential proteinase activated receptorSVERKER NYSTEDT*, KJELL EMILSSON*, CLAES WAHLESTEDTt, AND JOHAN SUNDELIN*t*Division of Molecular Neurobiology, The Wallenberg Laboratory, Lund University, P.O. Box 7031, S-220 07 Lund, Sweden; and tDivision of Neurobiology,Cornell University Medical College, New York, NY 10021

Communicated by Jan G. Waldenstrom, June 27, 1994 (received for review April 6, 1994)

ABSTRACT A DNA sequence encoding a G-protein-coupled receptor was isolated from a mouse genomic library.The predicted protein is similar in structure to the thrombinreceptor and has a similar activation mechanism. When ex-pressed inXenopus laevis oocytes, the receptor was activated bylow concentrations of trypsin (EC 3.4.21.4) and by a peptide(SLIGRL) derived from the receptor sequence, but was notactivated by thrombin (EC 3.4.21.5). Trypsin failed to activatea mutant receptor in which the presumed cleavage site Arg-34-Ser-35 was changed to an Arg-Pro sequence. The agonistpeptide (SLIGRL) activated equally well mutant and wild-typereceptors. Northern blot analysis demonstrated receptor tran-scripts in highly vascularized tissues such as kidney, smallintestine, and stomach. Because this, to our knowledge, is thesecond example, besides the thrombin receptor, of a proteolyt-ically activated seven-transmembrane G-protein-coupled re-ceptor, we have provisionally named it proteinase activatedreceptor 2.

Classically cell-surface receptors are activated by ligandbinding. This interaction induces a conformational change inthe receptor molecule that results in the signal being trans-mitted across the cell membrane. The activation is readilyreversible inasmuch as the ligand can dissociate from itsreceptor. Within the family of G-protein-coupled receptors adifferent mechanism of activation was recently revealed withthe cloning of the human thrombin receptor (1), whichappears to be activated as a result of proteolytic cleavage.For a long time it has been clear that the cellular effects of

thrombin and a number of other enzymes require them to beproteolytically active (see ref. 2). Two alternatives wereopen: either the enzyme cleaved its cell receptor directly, orsome other molecule was digested, releasing an unidentifiedactive factor. In their initial report Vu et al. (1) showed thatthe thrombin receptor belonged to the large family of seven-transmembrane pass receptors whose intracellular effects aremediated by heterotrimeric G proteins (3). This group alsodemonstrated that a synthetic peptide, corresponding to thenew amino terminus created by thrombin cleavage, potentlyactivated both platelets and the thrombin receptor expressedin Xenopus laevis oocytes. These observations have intro-duced another concept of receptor-agonist interaction. Aftercleavage the new receptor amino terminus functions as atethered ligand and interacts with some other hitherto unde-termined region of the receptor to effect signal transductionacross the membrane.

It has been proposed that not all the cellular effects ofthrombin can be ascribed to the newly cloned G-protein-coupled receptor (4), raising the possibility that there aresubtypes of thrombin receptors. The cloning of thrombinreceptors from Chinese hamster (5), rat (6), and mouse(GenBank accession no. L03529) has also been reported, butthese receptors are all very similar to the human receptor,

indicating that they represent the same kind of receptor indifferent species.When the human thrombin-receptor sequence was pub-

lished, it was also speculated that this would be the firstrepresentative of a class of proteolytically activated recep-tors. However, Southern blot experiments with genomicDNA to probe for similar genes have proved unsuccessful,and no other reports have yet come forth to validate thisspeculation. Still, given the profusion of serine proteinases invarious physiological systems-e.g., the blood-clotting cas-cade and the complement system, paired with the docu-mented cellular effects displayed by, among others, plasminand factor XIIa (7, 8), this failure seems rather to speak ofthelimitations of current methodologies.The results presented here will shed some light on these

issues. We report the cloning of a mouse genomic DNAsequence encoding a proteolytically activated receptor.§When expressed in frog oocytes, the receptor, which weprovisionally have named proteinase activated receptor 2(PAR-2), is activated by trypsin and by a synthetic peptidemade from the receptor sequence.

MATERIALS AND METHODSA mouse genomic library, cloned in the cosmid vector pTCF(9), was obtained from Rick A. Wetsel (Departments ofPediatrics and Microbiology and Immunology, WashingtonUniversity School of Medicine, St. Louis). Oligonucleotidesand synthetic peptides were obtained from The BiomolecularResource Facility of Lund University. Trypsin (bovine pan-creatic type III, EC 3.4.21.4) was purchased from Sigma, andhuman a-thrombin (EC 3.4.21.5) was from Johan Stenflo(Department ofClinical Chemistry, Malmo General Hospital,Malmd, Sweden).

Screening of a Mouse Genomic Library and DNA Sequenc-ing. Approximately 400,000 clones were screened by filterhybridization with a mixture oftwo 60-oligomers correspond-ing to transmembrane region two and six of the bovinesubstance K receptor (10). The probes and the screeningconditions were the same as described (11), except that alower temperature (60'C) was used during hybridization andwashings. Cosmid DNA was prepared from hybridizingclones and analyzed by Southern blotting. Selected frag-ments were subcloned in pBluescript (Stratagene), and bothstrands were sequenced by the dideoxynucleotide chain-termination method. DNA sequences were analyzed by usingthe University ofWisconsin Genetics Computer Group pack-age (12).Southrn Blot Analysis. Mouse genomic DNA (10 pg) was

cleaved with the restriction enzymes BamHI or Pst I, andfragments were separated on an agarose gel and blotted ontoa nylon membrane (Amersham Hybond-N). A fragment,encompassing the presumed PAR-2-coding region, was la-

Abbreviation: PAR-2, proteinase activated receptor 2.*To whom reprint requests should be addressed.§The sequence reported in this paper has been deposited in theGenBank data base (accession no. Z35158).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Pstl Xbal Clal Sall Pstl--b.-.*

-* -.

FIG. 1. Restriction map of the 3.7-kb Pst I fragment from cosPAR-2, including subcloning and sequencing strategy. The heavy linedenotes the presumed protein coding region of the PAR-2-encodinggene. Lines above the restriction map represent subcloned fragmentsthat were sequenced from the ends. The rest of the sequence wasobtained using specific primers, as indicated below by arrows.

beled by random priming and hybridized to the filter. Con-ditions were the same as for the colony hybridizations. Afterwashing in lx standard saline citrate (SSC)/0.1% SDS (lxSSC = 0.15 M NaCl/15 mM sodium citrate) at 600C the filterwas exposed to x-ray film for 2 days at -700C with anintensifying screen.

Northern Blot Analysis. Total tissue RNA was extractedfrom mouse tissues and separated on an agarose/formalde-hyde gel (13). The RNA was transferred to a nitrocellulosefilter (Amersham Hybond Extra), which was then baked for2 hr at 80'C. A PAR-2 probe (same as above) was hybridizedto the filter in 5 x SSC/5 x Denhardt's solution/salmon spermDNA at 50 pgmlh1/50% (vol/vol) formamide at 420C,washed in 0.1 x SSC/0.1% SDS at 60'C and exposed to a Fujiphosphoimaging screen.

Expression Constuts and Site-Directed Mutagenesis. Twooligonucleotide primers recognizing, respectively, the anti-sense and sense strands of the 5'- and 3'-ends of the receptorcoding sequence were used in a PCR reaction (94°C, 1 min;45°C, 1 min; 72°C, 1 min; 30 cycles) to generate a clonable

fragment encompassing only the coding region. The primers,designated L35 (5'-TCGGATCCACCATGTTCCATT-TAAAACACAG-3') and L36 (5'-TCGGATCCTCAGTAG-GAGGTTTTAACAC-3') had BamHI cleavage sites addedneartheir 5'-ends and in L35, the primer covering the initiatormethionine codon, an adenosine was substituted for guani-dine in position -3 respective to the initiatorATG to improvetranslation initiation. The PCR product was cloned inpBluescript, sequenced, and then cloned into the expressionvector pSG5 (14). In a control construct a point mutation atcodon number 35 (Ser-35 -* Pro) was generated by site-directed mutagenesis (15). Two bases were replaced in thatcodon to change the wild-type serine residue to a proline. Themutation was confirmed by nucleotide sequencing.

Expression in X. laevis Oocytes. Capped synthetic mRNA(cRNA) was prepared from expression constructs using aTransProbe kit from Pharmacia. The cRNA was dissolved inwater, and 34 nl (100 ng/t) was injected into X. laevisoocytes as described (16). Control oocytes were injected withsubstance K receptorcRNA or with water. The oocytes wereloaded with 45Ca2+ (100 ,uCi/ml; 1 Ci = 37 GBq), and theefflux of 45Ca2+ was measured as described (16).

RESULTSIsolation of a Genomic DNA Clone Ending a G-protein-

coupled Receptor. In a search for G-protein-coupled recep-tors we screened a mouse genomic library, from which thegenes for the mouse substance K and substance P receptorshave been cloned (11). The library was screened at moderatestringency with two oligonucleotides corresponding to two

5'CCCTGTCAGTCTTAAGATTCTAGAAGTCGCTGTCCTATACGGAACCCAAAACTCTCACTGTTAATGAAATACCATTGTCGGGGCGAAGATGTAGCTCAGTGGTAAAATACT -11TGCCAGCACACACAAGAATTAGACTTCAACCGTCACCAACTGCCCTGTGTAGGACGGTCGGTCACTGAAAGAGAATATTGTCTGCAATACTCTAATGACATCTGTCTGTGTTCATCTGAA -1

SP V1 MetPheHisLeuLysHisSerSerLeuThrValGlyProPheIleSerVaLMetIleLeuLeuArgPheLeuCysThrGlyArgAsnAsnSerLysGlyArgSerLeuIleGlyArgLeu

ATGTTCCATTTAAAACACAGCAGCCTTACTGTTGGACCATTTATCTCAGTAATGATTCTGCTCCGCTTTCTTTGTACAGGACGCAACAACAGTAAAGGAAGAAGTCTTATTGGCAGATTA 120

41 GluThrGlnProProIleThrGlyLysGlyValProValGluProGlyPheSerIleAspGluPheSerAlaSerIleLeuThrGlyLysLeuThrThrValPheLeuProValValTyrGAAACCCAGCCTCCAATCACTGGGAAAGGGGTTCCGGTAGAACCAGGCTTTTCCATCGATGAGTTCTCTGCGTCCATCCTCACCGGGAAGCTGACCACGGTCTTTCTTCCGGTCGTCTAC 240

IleIleValPheValIleGlyLeuProSerAsnGlyMetAlaLeuTrpIlePheLeuPheArgThrLysLysLySHisPrcII

)AlaValIleTyrMetAlaAsnLeuAlaLeuAlaAspLeu

III121 LeuSerValIleTrpPheProLeuLysIleSerTyrHisLeuHisGlyAsnAsnTrpValTyrGlyGluAlaLeuCysLysValLeuIleGlyPhePheTyrGlyArnMetTyrCysSer

CTCTCTGTCATCTGGTTCCCCCTGAAGATCTCCTACCACCTACATGGCAACAACTGGGTCTACGGGGAGGCCCTGTGCAAGGTGCTCATTGGCTTTTTCTATGGTAACATGTATTGCTCC 480

IV

IleLeuPheMetThrCysLeuSerValGlnArgTyrTrpValIleValAsnProMetGlyHisProArgLysLysAlaAsnIleAlaValGlyValSerLeuAlaIleTrpLeuLeuIleATCCTCTTCATGACCTGCCTCAGCGTGCAGAGGTACTGGGTGATCGTGAACCCCATGGGACACCCCAGGAAGAAGGCAAACATCGCCGTTGGCGTCTCCTTGGCAATCTGGCTCCTGATT

161600

201 PheLeuValThrIleProLeuTyrValMetLysGlnThrIleTyrIleProAlaLeuAsnIleThrThrCysHisAspValLeuProGluGluValLeuValGlyAspMetPheAsnTyrTTTCTGGTCACCATCCCTTTGTATGTCATGAAGCAGACCATCTACATTCCAGCATTGAACATCACCACCTGTCACGATGTGCTGCCTGAGGAGGTATTGGTGGGGGACATGTTCAATTAC 720

241 PheLeuSerLeuAlaIleGlyValPheLeuPheProAlaLeuLeuThrAlaSerAlaTyrValLeuMetIleLysThrLeuArgSerSerAlaMetAspGluHisSerGluLysLysArgTTCCTCTCACTGGCCATTGGAGTCTTCCTGTTCCCGGCCCTCCTTACTGCATCTGCCTACGTGCTCATGATCAAGACGCTCCGCTCTTCTGCTATGGATGAACACTCAGAGAAGAAAAGG 840

VI281 GlnArgAlaIleArgLeuIleIleThrValLeuAlaMetTyrPheIleCysPheAlaProSerAsnLeuLeuLeuValValHisTyrPheLeuIleLysThrGlnArgGlnSerHisVal

CAGAGGGCTATCCGACTCATCATCACCGTGCTGGCCATGTACTTCATCTGCTTTGCTCCTAGCAACCTTCTGCTCGTAGTGCATTATTTCCTAATCAAAACCCAGAGGCAGAGCCACGTC 960

VII

TyrAlaLeuTyrLeuValAlaLeuCysLeuSerThrLeuAsnSerCysIleA~pProPheValTyrTyrPheValSerLysAspPheArgAspHisAlaArgAsnAlaLeuLeuCysArgTACGCCCTCTACCTTGTCGCCCTCTGCCTGTCGACCCTCAACAGCTGCATAGACCCCTTTGTCTATTACTTTGTCTCAAAAGATTTCAGGGATCACGCCAGAAACGCGCTCCTCTGCCGA

3211080

361 SerValArgThrValAsnArgMetGlnIleSerLeuSerSerAsnLysPheSerArgLysSerGlySerTyrSerSerSerSerThrSerValLysThrSerTyrAGTGTCCGCACTGTGAATCGCATGCAAATCTCGCTCAGCTCCAACAAGTTCTCCAGGAAGTCCGGCTCCTACTCTTCAAGCTCAACCAGTGTTAAAACCTCCTACTGAGATGTCAAGCCTGCTTGATGATGATGATGATGATGGTGTGTGTGTG 3'

12001246

FIG. 2. Genomic nucleotide sequence from the mouse PAR-2 clone and predicted amino acid sequence of the large open reading frame.Amino acids are numbered at left, and nucleotides are numbered at right. The heavy overline denotes a putative signal peptide sequence, andthin overlines mark seven predicted transmembrane regions. The proposed serine protease cleavage site between Arg-34 and Ser-35 is indicatedby an arrowhead.

818 360

- - -

ATTATTGTGTTTGTGATTGGTTTGCCCAGTAATGGCATGGCCCTCTGGATCTTCCTTTTCCGAACGAAGAAGAAACACCCCGCCGTGATTTACATGGCCAACCTGGCCTTGGCCGACCTC

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transmembrane regions of the bovine substance K receptor.Among a large number of positive clones, one cosmid clone("cos PAR-2") has been characterized in some detail.Through restriction enzyme digestions and Southern blotanalyses the cosmid was shown to harbor a hybridizing3.7-kb Pst I fagment (Fig. 1). Sequence analyses of thisfragment, after additional subcloning and the use of specificprimers, revealed an open reading frame, putatively encodinga protein of 395 amino acid residues (Fig. 2). A hydropho-bicity plot of the deduced protein sequence showed sevensegments likely to form membrane-spanning regions and anamino-terminal hydrophobic stretch probably representing asignal peptide. Apart from the transmembrane segments, thededuced amino acid sequence contains a number of aminoacid residues generally conserved among G protein-coupledreceptors (3). These include, among others, Asn-91, Leu-115,Asp-119, Arg-171, and Pro-339. Other sequence features ofthe deduced protein are two potential sites for N-linkedglycosylation in the receptor extracellular regions (Asn-29and Asn-220) and several potential sites for phosphorylationby serine/threonine kinases in the third intracellular loop andthe intracellular carboxyl terminus.The Deduced Protein Sequence Is Similar to That of the

Thrombin Receptor. Searching the European Molecular Bi-ology Laboratory data base with the deduced protein se-quence of PAR-2 revealed the thrombin receptor (1, 5, 6) asits closest relative. The bovine substance K receptor se-quence, from which the probes were derived, displays verylimited similarity with the PAR-2 sequence outside the sec-ond transmembrane region. The mouse PAR-2 sequence hasan overall 30% identity to the human thrombin receptor (Fig.3) and 28% identity to the mouse thrombin receptor. Intransmembrane regions 44% of the amino acids are identicalbetween the two former receptors, and a few other stretchesare also well conserved; for instance in extracellular loop twothere are 10 consecutive identical amino acids. There areimportant differences, though. Except for the region wherethrombin cleaves the thrombin receptor (arrowhead in Fig.3), the amino-terminal part of PAR-2 has little in commonwith that of the human thrombin receptor. It is 29 amino acidresidues shorter and, most importantly, it does not possess astretch of acidic residues similar to the sequence in the

VPAR2 - MFHLKHBSLTVGPFISVMILLRFLCTGRNNSK LI G ETQ 43HSTHRR MGPRRLLLVAACF1LCGPLLSARTRARRPESKATNATLD FL PND 50

PAR2 PPI----------------------TGKGVPVEPGFidL4FSASIHiiGKL 71HSTHRR KYEPFWEDEEKNESGLTEYRLVSINKSSPLQKQLPAF1S DASG YUSSW 100

I II

PAR2 I I <Sl WINLF PA I 121HSTHRR GF S

II VVHILK 150

PAR2 -fI W o~ La*NVYlEAF KV LI G LRV-0 171HSTHRR FSV F k FgSELRFVTA MV 0 I0d 200

IV

PAR2 YWVIfN-GHPRKKANILVGV TIIFLVTIVMK YI 220HSTHRR FLA SLSWRTLG LASFT I AGVVLjVLKFIQAjQVNU 250

VPAR2 WT TCHVLPREV DMF <LSLAIG LF ALLTASA KLoiK 270HSTHRR JNVN VY YASAAMFCIF SIISFS ST 300

VIPAR2 DE ORI~pI fflAMY IC F LI- QRQSH 319HSTHRR SVANR--COFOOMCI FIA SFLSOSTTEA 348

VIIPAR2 CALODVAL PITVF jFVSKDFRDHARNAnLpRSVRTVNRMQ 369HSTHRR QF LC V CD OSCPLIB AfSECORYVYSC KESSDPSSYN 398

PAR2 I~LSSNKFSRKSGRYSSSSTS v SY-HSTHRR Sj QLMASKMDTCHSNLNNSIYk LLT

XSN

- 1.

- 1.'I

- 4 .Ati

FIG. 4. Southern blot of mouse genomic DNA digested witheither BamHI (left lane) or Pst I (right lane) hybridized to a probecovering the PAR-2 coding region. Positions of selected DNA sizemarkers (in kilobases) are shown at right.

thrombin receptor thought to mediate binding to the thrombinanion-binding exosite (17). The intracellular carboxyl-terminal sequence also shows very little similarity betweenthe two receptors.A PAR-2-Probe Recognizes a Single Fragment on Genomic

Southern Blots. To find out whether PAR-2 represented asingle-copy gene and also if probes prepared from it wouldrecognize the thrombin receptor gene in a hybridizationexperiment a Southern blot of mouse genomic DNA digestedwith eitherBamH1 orPst I was done. The blot was hybridizedto a probe corresponding to the whole coding part of PAR-2and washed at moderate strength. As shown in Fig. 4, onlya single band, corresponding to the expected PAR-2 frag-ment, is apparent in each lane.PAR-2 Is Expressed in Kidney, Small Intestine, Stomach,

and Eye. To clarify if the PAR-2 gene was expressed and, ifso, from its tissue distribution find clues as to the nature ofthe receptor it encoded, a Northern blot experiment wasdone. Ten micrograms each of total RNA from mouse eye,brain, skeletal muscle, heart, liver, stomach, small intestine,kidney, and testis was separated on an agarose/formalde-

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FIG. 3. Alignment ofthe predicted protein sequence ofthe mousePAR-2 with that of the human thrombin receptor (HSTHRR). Boxedamino acids are identical in the two receptors. Overlines indicate thepredicted transmembrane regions. The proposed cleavage sites arealigned and marked by an arrowhead. A few gaps have been insertedfor optimal alignment.

FIG. 5. Northern blot ofmouse tissue total RNA (10 jg per lane)hybridized to a probe recognizing the whole PAR-2 coding region.Tissue sources are given on the top, and positions of the ribosomalRNA bands are indicated at right. The single hybridizing species ineye, stomach, small intestine, and kidney corresponds to an mRNAof -3.0-kb size.

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hyde gel, transferred to a nitrocellulose membrane, andhybridized to the same probe as was used for the Southernblot. The result (Fig. 5) shows hybridization to a singletranscript of =3.0-kb size in kidney, small intestine, stomach,and eye. Although the amount of total RNA from each tissueseemed very similar as judged from a photograph of theethidium bromide-stained gel, a control hybridization with a,-actin probe resulted in variable signal intensities. Theresults, however, are consistent with data obtained withother techniques-i.e., reverse transcriptase-PCR and ribo-nuclease protection assays (data not shown).PAR-2 Is Activated by Trypsin and by a Receptor-Derived

Peptide. The similarity in sequence between PAR-2 and thethrombin receptor suggested that also PAR-2 could be acti-vated by proteolytic cleavage by an arginine-specific pro-teinase. To test this hypothesis cRNA encoding PAR-2,modified to improve translation as described in Materials andMethods, was injected in X. laevis oocytes, and efflux of45Ca2+ was measured after trypsin stimulation. Trypsin haspreviously been shown to activate the human thrombinreceptor (1). As controls, oocytes injected with either waterorcRNA encoding the mouse substance K receptor (16) wereused. Trypsin at 300 pM evoked a clear response that at 3 nMbecame comparable to the maximal response evoked bysubstance K on oocytes injected with substance K receptorcRNA (data not shown). Higher concentrations of trypsininevitably resulted in large amounts of 45Ca2+ flowing out ofthe cells, regardless of whether the cells expressed PAR-2 ornot.

If PAR-2 is a member of the family of proteolyticallyactivated receptors defined by the thrombin receptor, oneshould also expect that a peptide derived from the receptoramino terminus would activate the receptor. A peptide,corresponding to the six amino acid residues (SLIGRL)constituting the new amino terminus after a proposed cleav-age at Arg-34, was synthesized and used to stimulate oocytesexpressing PAR-2. In addition, the ability of the peptide andtrypsin to activate a mutant PAR-2, where Ser-35 had beenmutated to a proline residue (PAR-2 S35P), was investigated.The results, summarized in Fig. 6, show that the peptideSLIGRL at 100 ,uM activates both wild-type and mutantreceptors, whereas trypsin only activates the wild-typePAR-2. The EC50 of the agonist peptide was -5 ,uM, approx-imately three orders of magnitude higher than that of trypsin.

25

20

*:15

10 0 115

10

Wild type Mutant Water

FIG. 6. Agonist activity of trypsin (open bars) and activatingpeptide (SLIGRL; closed bars) onX. Iaevis oocytes expressing eitherwild-type PAR-2 or receptor with a mutated proteinase cleavage site(Ser-35 -- Pro). Control oocytes were injected with water instead ofcRNA. Oocytes were first challenged with trypsin (300 pM) followedby activating peptide (100 tM), and 45Ca2+ efflux was determined asdescribed (16). Data represent the means ± SEM of three parallelexperiments. Note that the responses to trypsin and activatingpeptide put together are approximately the same in wild-type andmutant receptors.

In contrast to trypsin, a-thrombin at concentrations up to 100nM failed to activate PAR-2.

DISCUSSIONThe cloning of a cellular thrombin receptor provided manyanswers long sought for concerning the mechanisms of cellactivation by thrombin. It gave the simplest, although per-haps somewhat unexpected, explanation to the problem whythe enzyme had to be proteolytically active to have any effecton cellular behavior. In analogy to what happens in proen-zyme conversion to active enzyme, but in contrast to theconventional model for receptor-ligand interactions, the re-ceptor itself had to be cleaved to transmit a signal across thecell membrane.From a mouse genomic library we have cloned a 3.7-kb

DNA fragment with an open reading frame putatively encod-ing a different proteinase-activated receptor-PAR-2. Thededuced protein sequence appears to represent a typicalmember of the rhodopsin family of G protein-coupled recep-tors. But because the genomic DNA sequence, as far as wehave determined it, does not reveal where transcriptionstarts, the definitive assignment of translation initiationcodon also will have to await the cloning of a PAR-2 cDNA.The sequence context of the proposed initiator methioninecodon is less than perfect for starting translation (18). Al-though we cannot at present exclude the possibility thattranslation starts at another methionine, there is no otherin-frame methionine codon further upstream, and the codon51 nt downstream, we believe, is improbable as initiatorbecause this would make the proposed signal peptide non-functional.As shown here, PAR-2 seems a close relative to the

thrombin receptor. A high overall similarity warrants akinship in evolutionary terms and, based upon the experi-ments done in the oocyte system, we conclude that PAR-2and the thrombin receptor share the same activation mech-anism. Thus, oocytes injected with cRNA encoding PAR-2are stimulated by trypsin and by a synthetic peptide(SLIGRL) made from the receptor sequence. Further evi-dence that proteolysis by trypsin is required for receptoractivation was obtained from experiments with a receptormutant, in which the presumed cleavage site Arg-34-Ser-35was mutated to the trypsin-resistant Arg-Pro sequence. Tryp-sin failed to activate the mutant receptor, whereas the agonistpeptide SLIGRL activated the mutant equally well as thewild-type receptor.

Trypsin activates PAR-2 at very low concentrations. Half-maximal response was seen at -1 nM, which is severalhundredfold lower than the concentration needed to activatethe thrombin receptor (1). Even at much lower concentra-tions than used by Vu et al. (1), trypsin, in our hands, evokedmassive efflux of 45Ca2+ also from uninjected oocytes. Thisdiscrepancy is probably due to differences in the preparationof the oocytes or different activities of the trypsin used.The agonist peptide (SLIGRL) has an EC50 (-5 ,.M) at

PAR-2, which is considerably higher than that of trypsin. Asimilar difference in efficacy is found also between thrombinand the thrombin-receptor agonist peptide (1). This mostlikely reflects the difference between a "tethered" ligand anda ligand free in solution, which through its carboxyl-terminalcharge also differs from the tethered ligand. An additionalmechanism that may explain part ofthe greater potency oftheenzyme is the possibility that a single enzyme moleculeactivates many receptors.The agonist hexapeptide derived from the mouse PAR-2

sequence, SLIGRL, is distinct from, but similar to, thethrombin-receptor activation peptide SFLLRN (human re-ceptor; SFFLRN in the hamster, mouse and rat sequences).The binding sites of the tethered ligands are not known, but

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the structural similarity of the two ligands suggests that alsothe binding sites are similar. From the PAR-2-thrombin-receptor sequence alignment (Fig. 3) it is clear that the regionshowing highest similarity is the second extracellular loop, aregion generally not conserved among G protein-coupledreceptors. The construction ofmutant and chimeric receptorsmay be useful to determine if this region plays any role inbinding of the ligands. Another issue in this context thatneeds to be addressed is whether an agonist peptide derivedfrom either of the receptors may also activate the otherreceptor.

It is not yet clear what enzyme is the endogenous activatorof PAR-2. The PAR-2 sequence lacks the 'acidic regionpresent in the thrombin receptor shown to be important forthrombin affinity (17), suggesting that PAR-2 is not a secondthrombin receptor. This view is supported by the fact that wefailed to activate PAR-2 with a-thrombin in the oocytesystem. We have not identified any sequence feature inPAR-2 that makes any proteinase a more likely candidatethan the other as activator of this receptor. We note thatPAR-2 is activated by trypsin at concentrations down to 300pM. The endogenous activator of PAR-2 may therefore bevery trypsin-like, if not trypsin itself, should the receptor beaccessible for trypsin.As judged from the Northern and Southern blots, there is

only one transcript from a single PAR-2 gene. Using PAR-2probes, we have not detected any cross-hybridizing tran-scripts or genomic fragments, even at low stringency. Toexpand, if possible, the family of protease receptors further,alternative approaches to cross-hybridization probably haveto be used.From the tissue distribution of PAR-2 mRNA it is difficult

to draw any conclusions about the physiological function ofPAR-2. The presence oftranscripts in kidney, small intestine,and stomach would be consistent with a role for PAR-2 inregulation of blood vessel tone and permeability, these or-gans being highly. vascularized. The cloned PAR-2 togetherwith the available receptor-activation peptide will be usefulreagents to determine the identity and the physiological roleof this protease-activated receptor, as well as provide toolsfor elucidating general mechanisms of proteinase receptorfunction.

We thank Dr. Rick Wetsel for the cosmid library, Dr. JohanStenflo for providing human thrombin, and Helena Aberg for excel-lent technical assistance. This work was supported by grants fromthe Swedish Medical Research Council (B94-13X-09467), the KingGustaf V's 80th Birthday Trust, the Alfred Osterlund Trust, and theMedical Faculty, Lund University.

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