(Received for publication, November 22, 1994)
From the
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.
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). ()
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.
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.
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.
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.
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.
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.
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z48043[GenBank].