Proteinase activated receptor 2: role of extracellular loop 2 for ligand-mediated activation

1 Rat proteinase-activated receptor-2 (PAR(2)) variants were stably expressed in rat KNRK cells: (a) wild-type (wt)-PAR(2); (b) PAR(2)PRR, with the extracellular loop 2 (EL-2) sequence P231E232E233-mutated to PRR and (c) PAR(2)NET, 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 PAR(2). Its activity was increased 5 fold in PAR(2)PRR, but it was inactive in PAR(2)NET. 3 In PAR(2)PRR, 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 PAR(2)NET highly homologous in EL-2 with proteinase-activated receptor-1 (PAR(1)), the potency of the PAR(1)-derived peptide, SFLLR-NH2, was reduced by 100 fold compared with wt-PAR, whereas the potency of the PAR(2)-derived AP, SLIGRL-NH2 was reduced 10 fold. In contrast, the potency of trypsin in PAR(2)NET was almost the same as in wt-PAR(2). 5 We conclude that the acidic EL-2 tripeptide, PEE, in PAR(2) 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 PAR(2) and (b) that SFLLR-NH2 may interact differently with PAR(2) than it does with PAR(1). 7 The differential reductions in the potencies of SLIGRL-NH2, compared with trypsin in the PAR(2)PRR and PAR(2)NET cell lines point to differences between the interactions of the trypsin-revealed tethered ligand and the free receptor-activating peptide with PAR(2).