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 (KNRK-rPAR(2)) was used to study the structure-activity relationships (elevated intracellular Ca(2+)) for a series of: 1) PAR(1)-derived receptor-activating ligands (PAR(1)-APs) [SFLLR (P5), SFLLR-NH(2) (P5-NH(2)), SFLLRNP (P7), SFLLRNP-NH(2) (P7-NH(2)), and TFLLR-NH(2) (TF-NH(2))] and 2) PAR(2)-derived-activating-peptides (PAR(2)-APs) [SLIGRL-NH(2) (SL-NH(2)), SLIGR-NH(2) (GR-NH(2)), and SLIGKV-NH(2) (KV-NH(2))]. The activities of the PAR-APs were compared with the PAR(2)-AP analog trans-cinnamoyl-Leu-Ile-Gly-Arg-Leu-Orn-NH(2) tc-NH(2)), which as a [(3)H]propionyl derivative ([(3)H]propionyl-tc-NH(2)) was used to develop a radioligand-binding assay for PAR(2). The relative potencies of the PAR-APs in the Ca(2+)-signaling assay were tc-NH(2) = SL-NH(2) > KV-NH(2) congruent with P5-NH(2) > GR-NH(2) > P7-NH(2) > P7 > P5 > TF-NH(2). The reverse sequence PAR-APs, LSIGRL-NH(2) (LS-NH(2)), LRGILS-NH(2) (LR-NH(2)), FSLLRY-NH(2) (FSY-NH(2)), and FSLLR-NH(2) (FS-NH(2)), as well as the Xenopus PAR(1)-AP TFRIFD-NH(2), were inactive. The relative biological potencies of the peptides were in accord with their ability to compete for the binding of [(3)H]propionyl-tc-NH(2) (tc-NH(2) = SL-NH(2) > GR-NH(2) congruent with P5-NH(2) > P5) to KNRK-rPAR(2) cells, whereas inactive peptides (FS-NH(2); LR-NH(2)) showed no appreciable binding competition. Our data therefore 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 activating rat PAR(2) parallel their ability to activate human PAR(1). The relative receptor-binding activities of the PAR-APs, although in general agreement with their relative biological activities, point to differences in the intrinsic receptor-activating activities between the several PAR-APs. The binding assay we have developed should prove of use for the further study of PAR(2)-ligand interactions.
Endocrine Research Group, The University of Calgary, Faculty of Medicine, Calgary, Alberta, Canada.
B
B
Al-Ani
M
M
Saifeddine
A
A
Kawabata
B
B
Renaux
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S
Mokashi
M D
MD
Hollenberg
D000818:Animals; D001667:Binding, Competitive; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D006801:Humans; D008024:Ligands; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology
D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; C120581:trans-cinnamoyl-Leu-Ile-Gly-Arg-Leu-Orn-NH2tc-NH2; D014357:Trypsin