©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Mouse Proteinase-activated Receptor-2 cDNA and Gene
MOLECULAR CLONING AND FUNCTIONAL EXPRESSION (*)

(Received for publication, November 22, 1994)

Sverker Nystedt Anna-Karin Larsson Helena Åberg Johan Sundelin (§)

From the Division of Molecular Neurobiology, The Wallenberg Laboratory, Lund University, S-220 07 Lund, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We have reported the cloning from mouse genomic DNA of a fragment encoding a G-protein-coupled receptor related to the receptor for the blood clotting enzyme thrombin. Like the thrombin receptor this receptor is activated by proteolytic cleavage of its extracellular amino terminus. Because the physiological agonist at the receptor was unknown, we provisionally named it proteinase-activated receptor 2 (PAR-2). Here we present a PAR-2 cDNA of 2729 nucleotides that differs from the published genomic sequence at the 5` end, including a part of the protein coding region. The differences do not affect the peptide sequence of the activating proteinase cleavage site proper, but may include amino acid residues important for enzyme-substrate recognition. Analysis of the PAR-2 gene structure showed that the cDNA 5` end is derived from a separate exon located about 10 kilobases away from the 3` exon. Results from a primer extension experiment indicate that transcription starts at a unique site around nucleotide -203 respective to the translation initiation ATG. Chinese hamster ovary cells transfected with either the PAR-2 cDNA or a construct made from the published PAR-2 genomic sequence responded with intracellular calcium mobilization to stimulation with 1 nM trypsin, 10 µM PAR-2-activating peptide (SLIGRL), or 1 µM thrombin receptor-activating peptide (SFLLRN). Untransfected cells responded only to stimulation with thrombin receptor activating peptide. Only transcripts corresponding to the PAR-2 cDNA could be detected in three mouse tissues examined.


INTRODUCTION

Serine proteinases are involved in physiological processes ranging from food digestion to blood clotting and tissue remodeling in growth and after injury. Research on the roles played by these enzymes has focused on their activities in protein degradation and as humoral factors. But many serine proteinases also have potent cellular effects. In a number of reports (see, for example, (1, 2, 3, 4) ) it has been shown that blocking the enzyme's proteolytic activity also abolishes the influence on cell behavior, and so these effects apparently do not result from an ordinary receptor-ligand interaction.

Complementary DNAs encoding a receptor for the blood clotting enzyme thrombin have been cloned from several mammalian species and from the frog Xenopus laevis ( (5, 6, 7, 8) and EMBL accession number L03529). We have reported the cloning from mouse genomic DNA of a receptor that can be activated by trypsin in concentrations down to 1 nM(9) . The physiological agonist at this receptor is still not known, and therefore we provisionally named it proteinase-activated receptor-2 (PAR-2). (^1)

PAR-2 and the thrombin receptor both belong to the large family of cell surface receptors that span the membrane seven times and couple to heterotrimeric G-proteins, and both are activated by proteolytic cleavage of their extracellular amino termini. The cleavage unmasks what has been termed a ``tethered ligand'' at the new amino terminus that interacts with some other region of the receptor(10, 11, 12, 13) . Both receptors can be activated by synthetic peptides made from the new amino terminus, and it has been shown that these peptides can be as short as 6 amino acids and still exhibit full agonist activity at their respective receptors(9, 11, 12) .

Because the published PAR-2 DNA sequence was derived from a genomic clone, it was difficult to determine if the protein it encoded corresponded to the protein made from a mature mRNA. Apparently it coded for a protein that was functional in Xenopus oocytes(9) , but the possibility remained that the native receptor amino terminus was encoded by a separate exon. Here we report a mouse PAR-2 cDNA, whose 5` end differs from that of the published PAR-2 sequence. We present evidence that this cDNA represents the clearly predominant, if not the only, PAR-2 gene transcript. We also show that the two possible PAR-2 variants have similar functional properties when expressed in stably transfected cells.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes were from Boehringer Mannheim and Life Technologies, Inc., and Taq polymerase was from Promega. Oligonucleotides were synthesized at the Biomolecular Resource Facility at Lund University. Nylon membranes and radioactive isotopes were purchased from Amersham Corp. All cell culture media and supplements were from Life Technologies, Inc. except the dialyzed fetal bovine serum, which was from HyClone. Trypsin (EC 3.4.21.4), Fura-2 acetoxymethyl ester (Fura-2 AM) and probenecid were from Sigma. Peptides were a gift from COR Therapeutics Inc., San Francisco, CA.

Exon Trap Experiments

A 3.7-kb PstI mouse genomic DNA fragment containing the published PAR-2 coding region (9) was cloned in the exon trap vector pET01 (14) and then transfected into COS-7 cells by the calcium-phosphate precipitate method(15) . After 72 h total RNA was extracted(15) , and 10 µg was used for cDNA synthesis with Superscript reverse transcriptase (Life Technologies, Inc.) and a primer derived from the 3` part of the PAR-2 sequence (5`- GGGAACAGGAAGACTCCA-3`). The cDNA was subjected to PCR with one primer from the pET01 vector and one from the PAR-2 sequence (5`-GGGGGAACCAGATGACA-3`). Amplified fragments were separated by agarose gel electrophoresis, extracted (Qiaex), and cloned in pCRII (Invitrogen).

Cloning of a Complementary DNA

Mouse stomach total RNA was isolated by the guanidinium isothiocyanate method (15) and poly(A) RNA purified on oligo(dT)-cellulose. An oligo(dT)-primed cDNA library was constructed in gt10 using EcoRI linkers and reagents from Amersham. A portion of the library (about 500,000 clones) was screened with a probe, labeled with [alpha-P]dCTP by random priming, encompassing the presumed mouse PAR-2 coding sequence(9) . Filter hybridizations were done in 5 times SSC, 5 times Denhardt's solution, 0.1% SDS, 50 µgbulletml salmon sperm DNA at 60 °C and washings in 1 x SSC, 0.1% SDS at 60 °C. One hybridizing clone was isolated and phage DNA prepared from a liquid lysate. The insert was excised with EcoRI and cloned in pBluescript (Stratagene).

DNA Sequencing

Nucleotide sequences were determined on both strands by cycle sequencing, using an Applied Biosystems 373A DNA sequencer. For sequence analysis the University of Wisconsin Genetics Computer Group software package (16) and the Geneworks 2.2 (Intelligenetics Inc.) were used.

Mapping of the Mouse PAR-2 Receptor Gene

Cos PAR-2 (9) cosmid DNA was characterized by restriction mapping. DNA fragments were separated by agarose gel electrophoresis, blotted onto nylon membranes, and hybridized with a labeled ClaI-EcoRI DNA fragment derived from the 5` end of the PAR-2 cDNA. Two EcoRI-SalI DNA fragments, one of 2.7 kb and one of 5.5 kb, were subcloned in pBluescript.

Ribonuclease Protection Assays

Total RNA was isolated as above from mouse small intestine, kidney, and stomach and from chinese hamster ovary cells transfected with either a mouse PAR-2 receptor cDNA expression construct or a mouse PAR-2 genomic DNA expression construct (see expression experiments). A 388-nucleotide-long ClaI-XbaI PAR-2 genomic DNA fragment was cloned in pBluescript II SK. Plasmid DNA was linearized with XbaI and cRNA transcripts prepared with T7 RNA polymerase and alpha-S-UTP using the Ambion Maxiscript kit. Full-length transcripts were purified on a denaturing polyacrylamide gel and 4times10^5 cpm of the probe was hybridized to 10 µg of total RNA/sample. Unhybridized probe was digested with a mixture of RNase A and RNase T1, and protected fragments were separated on a 6% polyacrylamide sequencing gel, which was dried and exposed to x-ray film.

Polymerase Chain Reaction Amplification of the cDNA 5` End

Two µg of mouse kidney mRNA was used as starting material with a 5`-RACE Amplifinder kit (Clontech). Oligonucleotide primers for first strand cDNA synthesis and for PCR amplification were complementary, respectively, to nucleotides 378-394 and 109-126 of the mouse PAR-2 cDNA. PCR products were separated by agarose gel electrophoresis, and amplified fragments, further identified by Southern blotting, were isolated and cloned in pCRII for sequencing.

Primer Extension Analysis

A 20-base oligonucleotide complementary to nucleotides -1 to 19 of the mouse PAR-2 cDNA sequence was end-labeled with [P]ATP. Ten µg of total RNA was mixed with 10^5 cpm of labeled oligonucleotide and precipitated with sodium acetate and ethanol. The precipitate was dissolved in 5 µl of 250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl(2), heated to 65 °C for 10 min, and left overnight at 30 °C. Reverse transcription was done at 42 °C for 90 min in 20 µl of 62 mM Tris-HCl, pH 8.3, 94 mM KCl, 3.75 mM MgCl(2), 10 mM dithiothreitol, 500 µM dNTP with 200 units of Superscript reverse transcriptase (Life Technologies, Inc.). Samples were then phenolextracted and precipitated with sodium acetate and ethanol before they were dissolved in loading buffer and separated on a denaturing polyacrylamide gel.

Expression in Chinese Hamster Ovary Cells

Two different expression constructs were employed. One was the mouse PAR-2 cDNA, cloned in the EcoRI site of the eukaryotic expression vector pSG5(17) . The other, a genomic PAR-2 construct in pSG5, is described in Ref 9. Chinese hamster ovary cells (CHO-DG44) were transfected by the calcium-phosphate precipitate method with a mixture of 10 µg of either expression construct and 1 µg of plasmid pSV2-DHFR. Transfectants were selected for in medium without nucleosides supplemented with dialyzed fetal calf serum. Cell clones were isolated, expanded, and then tested for responsiveness to mouse PAR-2 activating peptide (SLIGRL). To the medium of responsive clones methotrexate was added at 50 nM.

Intracellular Ca Measurements

Cells were seeded on gelatin-coated 32-mm glass coverslips and grown for 1-2 days to 40-95% confluence. They were loaded with 2 µM Fura-2/AM in 500 µl of culture medium for 30 min at 37 °C in the presence of 2.5 mM probenecid. After three washes in extracellular medium buffer (135 mM NaCl, 4.6 mM KCl, 1.2 mM MgCl(2), 1.5 mM CaCl(2), 11 mM glucose, 11 mM HEPES, pH 7.4), coverslips were mounted in a chamber on the stage of a Nikon inverted microscope fitted to a photometer (Photon Technology International). Measurements were done in extracellular medium buffer at ambient temperature, and agonists were added with a micropipette. Fluorescence images were obtained by alternate excitation at 340 and 380 nm, and the emitted light was measured at 510 nm. The slit was adjusted to allow measurements from a single cell.


RESULTS AND DISCUSSION

In the course of characterizing the mouse PAR-2 gene we performed an exon trap experiment(18) . We wanted to find out whether the PAR-2 gene consisted of more than one exon and, in that case, where the exon-intron boundaries were. COS cells were transfected with a genomic DNA fragment containing the presumed entire coding region of PAR-2 (9) cloned in the exon trap vector pET01. After reverse transcription of isolated RNA, PCR was used to amplify PAR-2 transcripts. Subsequent DNA sequence analysis of the cloned PCR fragment revealed a functional splice acceptor site within the postulated protein coding region (Fig. 1B). This meant that probably either part of the coding region was simply missing from the published PAR-2 DNA sequence or at least there were other splice variants.


Figure 1: A, nucleotide sequence and deduced protein sequence of the large open reading frame of the mouse proteinase activated receptor 2 cDNA (uppercase letters) with 5`-flanking sequence (145 nucleotides in lowercase letters) that was derived from genomic DNA. The transcription start site, as mapped by primer extension, is marked with an asterisk. Nucleotides before the translation initiation codon have been given negative numbers. Two consensus polyadenylation signals are underlined. Overlines mark seven predicted membrane-spanning regions, and an arrowhead points to the proposed activating proteinase cleavage site between Arg-38 and Ser-39. B, DNA sequences near splice sites with deduced protein sequence. Consensus splice donor and acceptor sequences are underlined, and the splice junctions are indicated by slash marks. The cDNA nucleotide sequence is written in uppercase and intronic sequence in lowercase letters. Amino acid translation of the 3` intronic sequence is also shown but in lowercase letters. This is the sequence previously thought to represent the receptor amino terminus. Nucleotides and amino acids are numbered as in the cDNA and its protein translation, respectively.



Isolation of a PAR-2 Complementary DNA

A mouse stomach complementary DNA library was constructed in gt10 and screened with a genomic DNA probe recognizing the coding region of PAR-2(9) . One hybridizing clone was isolated and sequenced. The cDNA thus obtained (Fig. 1A) is 2729 nucleotides long and has a large open reading frame of 1197 nucleotides. Flanking this open reading frame is a GC-rich (69%) 5` leader sequence of 72 bases and a 3`-untranslated region that ends with a consensus polyadenylation signal (AATAAA; (19) ) 15 nucleotides before a short poly(A) stretch. An alternative polyadenylation signal is located at residues 2148-2153. The 3` region contains a GT repeat sequence shortly downstream of the translation stop codon.

A comparison of the cDNA sequence with the published PAR-2 genomic sequence (9) confirmed that it represented a PAR-2 transcript. Codons 30 through 399 are identical and so are the 3`-untranslated regions as far as the genomic sequence has been determined. But 5` to codon 30 the two sequences are completely different. They diverge precisely at the splice site identified in the exon trap experiment, implying that the 5` end of the cDNA is not the product of a cloning artifact, but derives from another exon. Like the published PAR-2 amino terminus, the peptide sequence encoded by the 5` end of the cDNA appears to represent a signal peptide. The most likely signal peptidase cleavage site is between Thr-25 and Glu-26(20) . If this is correct there are 5 amino acid residues in the mature receptor protein which are not present in the published PAR-2 sequence. The differences do not involve the proposed activating proteinase cleavage site itself, but might be near enough to be important for the activating enzyme's ability to recognize its receptor substrate.

The PAR-2 Gene Is Divided in Two Exons

To reexamine the organization of the mouse PAR-2 gene, a nucleotide probe was prepared from the cDNA that spanned the putative splice site junction and which would thus recognize both of the proposed exons. In Southern blot experiments with mouse PAR-2 cosmid DNA, a 5` exon was mapped to a location about 10 kilobases upstream from the rest of the coding region (Fig. 2). A DNA fragment containing this exon was isolated and partly sequenced. The cDNA sequence was confirmed and 145 additional nucleotides of the 5` sequence determined (see Fig. 1A). A consensus splice donor site was found at the 3` boundary of the exon (Fig. 1B). A genomic fragment containing the 3` part of the cDNA was also isolated, and with a combination of nucleotide sequencing and PCR experiments, we confirmed that it consists of a single exon (data not shown). Downstream of the polyadenylation site we found the sequence TGTGTTTG (not shown), which resembles the consensus YGTGTTYY present at this site in many mammalian genes(21) . An unusual feature is that 14 out of the 16 adenosines in the cDNA poly(A) stretch are also found in the genomic sequence. We conclude that the PAR-2 gene spans more than 13 kb and appears to consist of at least two exons separated by about 10 kb of intronic sequence. Although the thrombin receptor gene organization has not yet been reported, published PCR results point to the presence of an intron somewhere near the 5` end of the human thrombin receptor DNA sequence(22) . We would predict that this intron is located at a site analogous to that in the PAR-2 gene, near the end of the signal peptide coding sequence.


Figure 2: Organization of the mouse proteinase-activated receptor 2 gene and its relationship to the cDNA. The upper line is a restriction map of the 30-kilobase cos PAR-2 cosmid where the thick segments represent the two exons. Indicated restriction sites are: BamHI (B), ClaI (C), EcoRI (E), and SalI (S). Not all EcoRI sites are shown. The box below represents the 2729-base PAR-2 cDNA. Its large open reading frame is marked by the stippled section.



Ribonuclease Protection Assays Detect Only a Transcript Corresponding to the PAR-2 cDNA

A ribonuclease protection assay experiment was designed to find out which one of the PAR-2 cDNA and the published PAR-2 genomic sequence represents the predominant transcript expressed. A complementary RNA probe was synthesized that spanned the exon-intron boundary of the 3` exon. As shown in Fig. 3, this probe protects a fragment doublet of about 100 nucleotides (i.e. up to the splice site) in RNA samples from three different mouse tissues as well as in control RNA from CHO cells transfected with the mouse PAR-2 cDNA. In the other control RNA sample, from cells transfected with a ``genomic'' PAR-2 construct, the probe protects the expected 181-base fragment but also one of 100 bases. The pSG5 expression vector contains an intron from the rabbit beta-globin gene located upstream from the cloning site and so the extra protected fragment can be explained by the presence of a chimeric transcript that arises from a splice reaction between a vector donor site and an insert acceptor site. Our interpretation of these results is that the cDNA represents the clearly predominant, if not the only, transcript from the PAR-2 gene. We do not know why the protected fragments appear as doublets, but it may be due to ``breathing'' of the RNA-RNA duplex during ribonuclease digestion.


Figure 3: A, picture showing the origin of the 388-nucleotide ClaI-XbaI DNA fragment used to construct a cRNA probe for ribonuclease protection assays. The restriction map is explained in the legend to Fig. 2. One XbaI (X) restriction site is also shown. The arrowhead points to the location of the splice acceptor site. B, autoradiogram showing the sizes of protected fragments from the ribonuclease protection assay. Lanes 1 and 2 contain digested and undigested probe, respectively. Lanes 3-7 contain RNase-treated samples of probe plus RNA from CHO cells expressing a genomic PAR-2 construct(3) , CHO cells expressing the PAR-2 cDNA(4) , and mouse small intestine(5) , kidney (6) , stomach(7) . The marker is a ddTTP sequencing reaction of a template with known sequence.



Further Investigations of the PAR-2 Transcript 5` End

A RACE experiment, using mouse kidney mRNA, was done with the 2-fold purpose of detecting splice variants and delineating the extent of the transcript 5` end(s). A Southern blot of the final PCR reaction was sequentially hybridized to two probes specific for the two different 5` ends. Only the probe directed against the cDNA 5` end hybridized to the PCR product (not shown). This means that, at least in kidney, a transcript corresponding to the published PAR-2 sequence, if present at all, represents a very minor share of the PAR-2 protein produced. The PCR product was ligated to pCRII and a number of clones isolated and sequenced. Thus we were able to add 27 nucleotides to the 5` end of the PAR-2 cDNA.

A primer extension experiment was done with a primer complementary to nucleotides surrounding the cDNA initiator ATG. A unique transcription start site around nucleotide -203 respective to the translation initiation codon was detected in RNA from mouse stomach and small intestine (Fig. 4). That is about 104 nucleotides further up from the start site indicated in the RACE experiment. This discrepancy is probably most easily explained by the fact that reverse transcription was carried out under different conditions in the two experiments and therefore differentially susceptible to premature termination due to mRNA secondary structure.


Figure 4: Primer extension experiment to define the PAR-2 transcription start site. The same primer, complementary to a sequence around the cDNA translation initiation codon, was used in reverse transcription and to generate the marker sequence ladder. A single extension product (arrowhead) was detected in RNA samples from mouse stomach (lane 1) and small intestine (lane 2). No transcripts were detected in RNA from heart (3) . The size of the extension products corresponds to a transcription start site around nucleotide -203 respective to the translation start ATG.



Cells Transfected with Different PAR-2 Constructs Respond Similarly

An expression construct made from the published genomic sequence has been shown to confer responsiveness to trypsin and PAR-2-activating peptide (PAR-2 AP; SLIGRL) to Xenopus oocytes. We wanted to test if both this construct and the cDNA could be expressed in stably transfected cells and, if so, look for functional differences in the receptors produced. Chinese hamster ovary cells were transfected and stable cell lines established. Cells were loaded with Fura-2 and changes in intracellular calcium concentration measured after stimulation with PAR-2 AP at 10 µM followed by thrombin receptoractivating peptide (TRAP; SFLLRN) at 1 µM or 1 nM trypsin followed by TRAP (n geq 2). As shown in Fig. 5, cells expressing the different receptors responded in essentially the same way to these substances. Cells transfected with either construct responded to PAR-2 AP, trypsin, and to TRAP with a transient increase in intracellular calcium concentration, whereas untransfected cells responded clearly only to TRAP. This is expected, as CHO cells express thrombin receptors endogenously. Some untransfected cells also responded to trypsin with a small and slowly developing elevation in calcium concentration.


Figure 5: Recordings of [Ca] responses in single chinese hamster ovary cells. Cells were transfected with the mouse PAR-2 genomic construct (A and D), the mouse PAR-2 cDNA (B and E), or untransfected (C and F). The arrowheads denote the addition of the following reagents: A-C, 1 nM trypsin (first arrow) and 1 µM TRAP (second arrow); D-F, 10 µM PAR-2 AP (first arrow) and 1 µM TRAP (second arrow).



One thing to bear in mind, when looking at these recordings, is that they represent responses of single cells and therefore inherently variable. We therefore cannot say anything about quantitative differences in the responses of the two receptor variants, only that they readily respond to the same substances. No transcript like the PAR-2 ``genomic'' sequence was detected in ribonuclease protection assays or in 5`-RACE experiments. Nevertheless, it is still possible that such a transcript is expressed in a limited tissue subset or during a certain period of animal development. If so, it encodes a functional receptor.

In addition to the presence of the sequence encoding an apparently functional signal peptide in front of the second exon, there is another peculiarity about the PAR-2 gene: 14 of the adenosines in the cDNA poly(A) tail are found also in the genomic sequence. This feature is one of the characteristics of nonviral retroposons (23) and so it may hold a clue as to how the PAR-2 gene evolved. Can it be that this short stretch of adenosines belonged to an mRNA from the ancestral proteinase receptor, which was accidentally reverse-transcribed and integrated at a new site in the genome? There is at least one example described of such an event giving rise to a functional new gene(24) . Whether these two unusual features have any bearing upon the function and origin of the PAR-2 may be resolved through characterization of additional proteinaseactivated receptor genes.


FOOTNOTES

*
This work was supported by the Swedish Medical Research Council (B94-13X-09467), the Gustaf V's 80th Birthday trust, the Alfred Österlund Trust, the Medical Faculty, Lund University, and COR Therapeutics Inc., San Francisco, CA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z48043[GenBank].

§
To whom correspondence should be addressed: Division of Molecular Neurobiology, The Wallenberg Laboratory, Lund University, P. O. Box 7031, S-220 07 Lund, Sweden. Tel.: 46-46-104298; Fax: 46-46-104324; johan.sundelin{at}wblab.lu.se.

(^1)
The abbreviations used are: PAR-2, proteinase-activated receptor 2; PAR-2 AP, proteinase-activated receptor 2-activating peptide; TRAP, thrombin receptor-activating peptide; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; CHO, Chinese hamster ovary.


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