THE MOUSE PROTEINASE-ACTIVATED RECEPTOR-2 CDNA AND GENE - MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION

We have reported the cloning from mouse genomic DNA of a fragment enco ding a G-protein-coupled receptor related to the receptor for the bloo d clotting enzyme thrombin. Like the thrombin receptor this receptor i s activated by proteolytic cleavage of its extracellular amino terminu s, Because the physiological agonist at the receptor was unknown, we p rovisionally named it proteinase-activated receptor 2 (PAR-2). Here we present a PAR-2 cDNA of 2729 nucleotides that differs from the publis hed genomic sequence at the 5' end, including a part of the protein co ding region. The differences do not affect the peptide sequence of the activating proteinase cleavage site proper, but may include amino aci d residues important for enzyme-substrate recognition. Analysis of the PAR-2 gene structure showed that the cDNA 5' end is derived from a se parate 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 ATC, Chinese hamster ovary cells transfected with either th e PAR-2 cDNA or a construct made from the published PAR-2 genomic sequ ence responded with intracellular calcium mobilization to stimulation with 1 nM trypsin, 10 mu M PAR-2-activating peptide (SLIGRL), or 1 mu M thrombin receptor-activating peptide (SFLLRN). Untransfected cells r esponded only to stimulation with thrombin receptor activating peptide . Only transcripts corresponding to the PAR-2 cDNA could be detected i n three mouse tissues examined.