title: Proteinase activated receptor 2: Role of extracellular loop 2 for ligand-mediated activation.
abstract: 1. Rat proteinase-activated receptor-2 (PAR2) variants were stably expressed in rat KNRK cells: (a) wild-type (wt) - PAR2; (b) PAR2PRR, with the extracellular loop 2 (EL-2) sequence P231E232E233mutated to PRR and (c) PAR2NET, with the EL-2 sequence, PEEV changed to NETL. Cell lines were evaluated for their sensitivity (calcium signalling) towards trypsin and the receptor-activating peptides, SLIGRL-NH2, SLIGEL-NH2, trans-cinnamoyl(tc)-LIGRLO-NH2, and SFLLR-NH2. 2. SLIGEL-NH2 exhibited low potency (1 : 200 relative to SLIGRL-NH2) in wild-type PAR2. Its activity was increased 5 fold in PAR2PRR, but it was inactive in PAR2NET. 3. In PAR2PRR, the potencies of SLIGRL-NH2, tc-LIGRLO-NH2, and SFLLR-NH2 were decreased by 80 - 100 fold. But, the potency of trypsin was decreased by only 7 fold. 4. In PAR2NET, highly homologous in EL-2 with proteinase-activated receptor-1 (PAR1), the potency of the PAR1-derived peptide, SFLLR-NH2, was reduced by 100 fold compared with wt-PAR2, whereas the potency of the PAR2-derived AP, SLIGRL-NH2 was reduced 10 fold. In contrast, the potency of trypsin in PAR2NET was almost the same as in wt-PAR2. 5. We conclude that the acidic EL-2 tripeptide, PEE, in PAR2 plays an important role in governing agonist activity. 6. The data obtained with the PEEV-->NETL mutation suggested: (a) that SLIGRL-NH2 and SFLLR-NH2 interact in a distinct manner with PAR2 and (b) that SFLLR-NH2 may interact differently with PAR2 than it does with PAR1. 7 The differential reductions in the potencies of SLIGRL-NH2, compared with trypsin in the PAR2PRR and PAR2NET cell lines point to differences between the interactions of the trypsin-revealed tethered ligand and the free receptor-activating peptide with PAR2.
authors.affiliation: Endocrine Research Group, University of Calgary, Faculty of Medicine, Calgary, AB, Canada T2N 4N1.
authors.forename: B
authors.initials: B
authors.lastname: Al-Ani
authors.forename: M
authors.initials: M
authors.lastname: Saifeddine
authors.forename: A
authors.initials: A
authors.lastname: Kawabata
authors.forename: M D
authors.initials: MD
authors.lastname: Hollenberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001711:Biotransformation / Q000187:drug effects / Q000235:genetics; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D005110:Extracellular Space / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D051381:Rats; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin / Q000819:agonists* / Q000235:genetics* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; D014357:Trypsin