Binding of a highly potent protease‐activated receptor‐2 (PAR2) activating peptide, [³H]2‐furoyl‐LIGRL‐NH₂, to human PAR2

Abstract

To determine the binding characteristics of a highly potent agonist for protease‐activated receptor‐2 (PAR2), 2‐furoyl‐Leu‐Ile‐Gly‐Arg‐Leu‐amide (2‐furoyl‐LIGRL‐NH2), whole‐cell binding assays were performed utilising a radioactive ligand, [3H]2‐furoyl‐LIGRL‐NH2. Specific binding of [3H]2‐furoyl‐LIGRL‐NH2 was observed in NCTC2544 cells, dependent upon PAR2 expression, and competitively displaced by the addition of unlabeled PAR2 agonists. Scatchard analysis of specific saturation binding suggested a single binding site, with Kd of 122±26.1 nM and a corresponding Bmax of 180±6 f mol in 3.0 × 105 cells. The relative binding affinities of a series of modified PAR2 agonist peptides obtained from competition studies paralleled their relative EC50 values for Ca2+ mobilisation assays, indicating improved binding affinities by substitution with 2‐furoyl at the N‐terminus serine. Pretreatment of cells with trypsin reduced specific binding of [3H]2‐furoyl‐LIGRL‐NH2, demonstrating direct competition between the synthetic agonist peptide and the proteolytically revealed tethered ligand for the binding site of the receptor. In HCT‐15 cells endogenously expressing PAR2, the binding of [3H]2‐furoyl‐LIGRL‐NH2 was displaced by addition of unlabeled ligands, Ser‐Leu‐Ile‐Gly‐Lys‐Val (SLIGKV‐OH) or 2‐furoyl‐LIGRL‐NH2. The relative binding affinity of 2‐furoyl‐LIGRL‐NH2 to SLIGKV‐OH was comparable to its relative EC50 value for Ca2+ mobilisation assays. The binding assay was successfully performed in monolayers of PAR2 expressing NCTC2544 and human umbilical vein endothelial cells (HUVEC), in 96‐ and 24‐well plate formats, respectively. These studies indicate that [3H]2‐furoyl‐LIGRL‐NH2 binds to human PAR2 at its ligand‐binding site. The use of this radioligand will be valuable for characterising chemicals that interact to PAR2. British Journal of Pharmacology (2005) 145, 255–263. doi:

Derivatized 2-Furoyl-LIGRLO-amide, a Versatile and Selective Probe for Proteinase-Activated Receptor 2: Binding and Visualization

“…7, histogram). Our new data complement and support a study that appeared upon completion of our work (Kanke et al, 2006), demonstrating the utility of a 3 H-labeled derivative of 2-furoyl-LIGRL-NH 2 to serve as a ligand binding reagent for PAR 2 . Our data indicate that the affinity and selectivity of the ornithine-substituted derivatives (both tritiated and labeled with Alexa Fluor 594) are essentially equivalent to those of 3 H-labeled 2-furoyl-LIGRL-NH 2 .…”

“…2), indicating a lower capacity binding site with a somewhat higher affinity (approximately 50 nM) than the one previously reported (120 nM) (Kanke et al, 2006). The higher affinity site we report here was not observed by Kanke et al, (2006). Our finding of curvilinearity of the Scatchard plot at the extremes of the concentration range that we used was not explored further.…”

“…2) reveals a plateau in binding in the middle of the curve (between approximately 50 and 120 nM: Fig. 2), indicating a lower capacity binding site with a somewhat higher affinity (approximately 50 nM) than the one previously reported (120 nM) (Kanke et al, 2006). The higher affinity site we report here was not observed by Kanke et al, (2006).…”

Kallikrein-Related Peptidase 14 Acts on Proteinase-Activated Receptor 2 to Induce Signaling Pathway in Colon Cancer Cells

“…Several lines of evidence indicate that KLK14 signals in HT29 cells only through PAR-2 and not via PAR-1: i) HT29 cells challenged with thrombin, the PAR-1 agonist, did not attenuate the KLK14-induced Ca 2ϩ flux; ii) desensitization of PAR-2 with 2-furoyl-LIGRLO-NH 2, a potent PAR-2-specific AP, 27 abrogated all KLK4-induced calcium transients while the response to PAR-1-specific peptide remained unaffected; and iii) KLK14 specifically induced PAR-2 internalization without any effect on PAR-1 surface localization. These results are in agreement with those of two others studies showing that KLK14, KLK5, and KLK6 are strong activators of PAR-2 in cells recombinantly expressing these receptors.…”

“…Reagents were obtained from the following sources: the APs TFLLR-NH 2 (AP1, a PAR-1 agonist) and SLIGKV-NH 2 (AP2, a PAR-2 agonist) or SFLLRN-NH 2 [the thrombin receptor agonist peptide (TRAP) that activates PAR-1 and PAR-2] and 2-furoyl-LIGRLO-NH 2 (a potent PAR-2 agonist) 27 from NeoMPS (Strasbourg, France); purified recombinant KLK14 (2 nmol/L of KLK14 equivalent to 1 U/mL of trypsin-like activity) has been previously described 28 ; highly purified ␣-thrombin (3000 U/mg) from Kordia Laboratory Supplies (Leiden, the Netherlands); trypsin (16,000 U/mg) and Alexa Fluor 488 dye-conjugated goat anti-mouse antibody from Life Technology Inc. (Cergy Pontoise, France); and Fura-2/AM from Molecular Probes (Leiden). Antibodies were purchased from the following vendors: phospho-specific antibodies to ERK1/2 from Cell Signaling Technologies (Beverly, MA) and polyclonal anti-ERK1/2 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA).…”