Proteinase-activated receptor 2: differential activation of the receptor by tethered ligand and soluble peptide analogs.
Activation of rat proteinase-activated receptor 2 (PAR2) by trypsin involves the unmasking of the tethered sequence S(37)LIGRL(42) that either tethered or on its own as a free peptide, activates PAR2. We aimed to determine whether different peptide sequences acting either as trypsin-revealed tethered ligands or as soluble peptides had the same relative activities for triggering the receptor. A comparison was also made between the different soluble and tethered receptor activating sequences in receptor constructs with extracellular loop 2 (ECL2) residues E(232)E(233) (PAR2SR/EE) mutated to R(232)R(233) (PAR2SR/RR). Using site-directed mutagenesis, we prepared PAR2 constructs with trypsin-revealed tethered ligand sequences corresponding to the synthetic receptor-activating peptides (PAR2APs): SLIGRL-NH(2) (SR-NH(2)), SLIGAL-NH(2) (SA-NH(2)), and SLIGEL-NH(2) (SE-NH(2)). Kirsten virus-transformed rat kidney cells stably expressing 1) wild-type PAR2 with site-mutated tethered ligands (PAR2SA/EE and PAR2SE/EE); 2) wild-type PAR2 with ECL2 mutated to R(232)R(233) (PAR2SR/RR); and 3) PAR2 constructs with both the RR mutation in ECL2 and a mutation in the tethered ligand (PAR2SA/RR and PAR2SE/RR) were assessed for receptor-mediated calcium signaling and cell growth inhibition, upon activation either by trypsin or the above-mentioned PAR2APs. Trypsin exerted equivalent and full agonist activity on the PAR2 constructs, causing a maximum response between 20 to 80 nM. In contrast, the PAR2APs as free peptide agonists showed marked potency differences in all wild-type receptors with mutated tethered ligands (SR-NH(2) >> SA-NH(2) >> SE-NH(2)) and in all ECL2 RR mutated constructs (SE-NH(2) > SR-NH(2) >> SA-NH(2)). We conclude that for receptor activation, the trypsin-revealed PAR2 tethered ligand sequence interacts differently for receptor activation than does the same peptide sequence as a free peptide.
Al-Ani
Bahjat
B
Diabetes and Endocrine Research Group, Departments of Pharmacology and Therapeutics, University of Calgary Health Sciences Centre, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. alani@ucalgary.ca
Wijesuriya
Suranga J
SJ
Hollenberg
Morley D
MD
D019943:Amino Acid Substitution; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D007150:Immunohistochemistry; D008024:Ligands; D021382:Protein Sorting Signals / Q000502:physiology*; D051381:Rats; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin / Q000187:drug effects* / Q000235:genetics; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology
D017931:DNA Primers; D018076:DNA, Complementary; D008024:Ligands; D021382:Protein Sorting Signals; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; D014357:Trypsin