Proteinase-activated receptor 2 (PAR(2)): Development of a ligand-binding assay correlating with activation of PAR(2) by PAR(1)- and PAR(2)-derived peptide ligands

A cloned rat proteinase-activated receptor (PAR)(2)-expressing cell line (K NRK-rPAR(2)) was used to study the structure-activity relationships (elevat ed intracellular Ca2+) for a series of: 1) PAR(1)-derived receptor-activati ng ligands (PAR(1)-APs) [SFLLR (P5), SFLLR-NH2 (P5-NH2), SFLLRNP (P7), SFLL RNP-NH2 (P7-NH2), and TFLLR-NH2 (TF-NH2)] and 2) PAR(2)-derived-activating- peptides (PAR(2)-APs) [SLIGRL-NH2 (SL-NH2), SLIGR-NH2 (GR-NH2), and SLIGKV- NH2 (KV-NH2)]. The activities of the PAR-APs were compared with the PAR(2)- AP analog trans-cinnamoyl-Leu-Ile-Gly-Arg-Leu-Orn-NH2 tc-NH2), which as a [ H-3]propionyl derivative ([H-3]propionyl-tc-NH2) was used to develop a radi oligand-binding assay for PAR(2). The relative potencies of the PAR-APs in the Ca2+-signaling assay were tc-NH2 = SL-NH2 > KV-NH2 congruent to P5-NH2 > GR-NH2 > P7-NH2 > P7 > P5 > TF-NH2. The reverse sequence PAR-APs, LSIGRL- NH2 (LS-NH2), LRGILS-NH2 (LR-NH2), FSLLRY-NH2 (FSY-NH2), and FSLLR-NH2 (FS- NH2), as well as the Xenopus PAR(1)-AP TFRIFD-NH2, were inactive. The relat ive biological potencies of the peptides were in accord with their ability to compete for the binding of [H-3]propionyl-tc-NH2 (tc-NH2 = SL-NH2 > GR-N H2 congruent to P5-NH2 > P5) to KNRK-rPAR(2) cells, whereas inactive peptid es (FS-NH2; LR-NH2) showed no appreciable binding competition. Our data the refore validate a ligand-binding assay for the use in studies of PAR(2) and indicate that the relative biological potencies of the PAR(1)-APs for acti vating rat PAR(2) parallel their ability to activate human PAR(1). The rela tive receptor-binding activities of the PAR-APs, although in general agreem ent with their relative biological activities, point to differences in the intrinsic receptor-activating activities between the several PAR-APs. The b inding assay we have developed should prove of use for the further study of PAR(2)-ligand interactions.