The Mouse Proteinase-activated Receptor-2 cDNA and Gene

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 microM PAR-2-activating peptide (SLIGRL), or 1 microM 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.

Molecular cloning, expression and potential functions of the human proteinase-activated receptor-2

“…RNA probes were transcribed from plasmids containing a 1.2 kb fragment comprising most of the coding region of mouse PAR2 and a 0.8 kb BspEI-ClaI fragment of the mouse thrombin receptor coding region. The mouse PAR2 fragment was cloned by PCR from mouse genomic DNA by using primers, 5h-CGTAAAGGA-AGAAGTCTTATTGGC-3h and 5h-CACATCCTCAGGTAC-AGCTCAGTA-3h, based on published sequences [4,5]. Sense and antisense probes were transcribed from linearized plasmids by using T3 or T7 RNA polymerases with $&S-labelled UTP.…”

“…The ATG codon was surrounded by a consensus sequence for initiation of translation [12]. The reading frame encoded a protein of 397 residues (calculated M r 44125) with seven hydrophobic, presumably membrane-spanning, domains and an N-terminal hydrophobic region that is compatible with a signal peptide with the most likely cleavage site between Thr#& and Ile#' [5,13]. Putative signal peptides in the G-protein coupled receptor family have been found only in proteinase-activated receptors and those with long N-terminal tails, such as the thyrotropin receptor [14].…”

“…for AP, 18 µM) ( Figures 7A and 8A). Similarly, thrombin and trypsin are more potent activators of the thrombin receptor and mouse PAR-2 respectively than activating peptides corresponding to the new N-terminus [2,4,5]. Presumably the tethered ligand is maintained at a high concentration and is favourably presented to the binding sites by its physical attachment to the receptor, which it can activate with high affinity.…”

“…Thrombin cleaves this receptor within the extracellular N-terminus at LDPR SFLL, forming an N-terminal tethered ligand that activates the receptor. The second member of this family is PAR-2, recently cloned from a mouse genomic library [4,5]. Trypsin cleaves the N-terminus of PAR-2 at SKGR SLIG, forming a tethered ligand.…”

“…Trypsin cleaves the N-terminus of PAR-2 at SKGR SLIG, forming a tethered ligand. Peptides corresponding to the tethered ligand sequences of the thrombin receptor and PAR-2 are full agonists and thus can be used to investigate receptor functions [2][3][4][5].…”