The Mouse Proteinase-activated Receptor-2 cDNA and Gene
MOLECULAR CLONING AND FUNCTIONAL EXPRESSION (*)
- From the Division of Molecular Neurobiology, The Wallenberg Laboratory, Lund University, S-220 07 Lund, Sweden
- § 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
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.
Footnotes
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↵* 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].
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↵1 The abbreviations used are:
- PAR-2
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proteinase-activated receptor 2
- PAR-2 AP
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proteinase-activated receptor 2-activating peptide
- TRAP
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thrombin receptor-activating peptide
- PCR
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polymerase chain reaction
- RACE
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rapid amplification of cDNA ends
- CHO
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Chinese hamster ovary.
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- Received November 22, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
