PAR2
1. Protease-Activated Receptor 2 (PAR2, G protein-coupled receptor, Ki = 320 nM for competitive displacement of [³H]2-furoyl-LIGRL-NH₂ from human PAR2; EC50 = 450 nM for PAR2-mediated calcium mobilization in transfected cells) [1]

The peptides that activate PAR2 are SLIGKV-OH, SLIGRL-OH, SLIGKV-NH₂, and SLIGRL-NH₂. Synthetic agonist peptides that mimic the tethered ligand of PAR2, Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH), Ser-Leu-Ile-Gly-Arg-Leu (SLIGRL-OH), and their amidated forms Ser-Leu-Ile-Gly-Lys-Val-amide (SLIGKV-NH₂) Because Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH₂) has been shown to be able to activate the receptor without enzymatic cleavage, it has been used as a biological tool to investigate the physiological functions of PAR2. Among the four family subgroups of G-protein-coupled receptors (GPCRs), or PARs, is protease-activated receptor-2, amide. Protease-activated receptors are unique among GPCRs in that they are activated by a proteolytic mechanism. Trypsin, tryptase, and coagulation factors VIIa and Xa are examples of activating proteases for PAR2 that cleave a particular extracellular amino-terminal domain of the receptor to reveal a "tethered ligand," SLIGKV- for human PAR2 and SLIGRL- for mouse/rat PAR2, respectively. This ligand then interacts with the receptor's activation domain to start intracellular signaling pathways[1]. Protease-activated receptor-2 (PAR2) is expressed in a broad range of human tissues and cells and has been linked to the pathophysiology of multiple inflammatory and autoimmune disorders. Proteolysis activates a family of seven transmembrane domain receptor proteins, including PAR2. The process of enzymatic digestion reveals an N-terminus ligand sequence, which binds intramolecularly to the activation site on the extracellular loop II. This triggers nuclear factor-kappa B (NF-κB)-regulated gene transcription and a G-protein-mediated cell-signalling cascade[2].

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1. PAR2 receptor binding activity: Protease-Activated Receptor-2, amide (SLIGKV-NH₂) exhibited concentration-dependent competitive binding to human PAR2 in membrane preparation assays. It displaced the radiolabeled PAR2 agonist [³H]2-furoyl-LIGRL-NH₂ from PAR2 with a Ki value of 320 nM, indicating moderate binding affinity for the receptor’s activation site. The compound showed no significant binding to other protease-activated receptors (PAR1/PAR3/PAR4) at concentrations up to 10 μM (residual binding > 90% for off-target PAR subtypes) [1]
2. PAR2-mediated signaling activation in dendritic cells (DCs): In murine bone marrow-derived dendritic cells (BMDCs), Protease-Activated Receptor-2, amide (SLIGKV-NH₂) (100 nM–5 μM) dose-dependently triggered PAR2 signaling cascades. At 1 μM, it increased phosphorylation of ERK1/2 (2.3-fold relative to control) and p38 MAPK (1.8-fold relative to control) within 15 min of stimulation. The compound also enhanced DC antigen uptake (FITC-dextran internalization increased by 41% at 1 μM) and upregulated costimulatory molecule expression (CD86 and MHC II levels elevated by 35% and 28% respectively at 1 μM) [2]
3. T-cell activation promotion via DC priming: In in vitro DC-T cell coculture systems, BMDCs pretreated with Protease-Activated Receptor-2, amide (SLIGKV-NH₂) (1 μM) for 24 h induced a 2.7-fold increase in CD4⁺ T-cell proliferation (assessed via [³H]thymidine incorporation) and a 3.1-fold elevation in IFN-γ secretion (measured via ELISA) compared with untreated DCs, confirming its ability to enhance DC-mediated T-cell activation [2]

1. Dendritic cell antigen transport and T-cell activation in mice: In C57BL/6 mice, subcutaneous administration of Protease-Activated Receptor-2, amide (SLIGKV-NH₂) (500 μg/kg) at the site of ovalbumin (OVA) antigen injection significantly promoted DC migration from peripheral tissues to draining lymph nodes (DLNs). At 48 h post-administration, the number of OVA-loaded DCs in DLNs was 2.5-fold higher than in vehicle-treated controls. The compound also enhanced OVA-specific CD4⁺ T-cell responses in DLNs: OVA-specific T-cell proliferation increased by 3.2-fold, and IFN-γ-producing CD4⁺ T-cell frequency rose from 4.2% (control) to 12.8% (compound-treated), with no significant effect on CD8⁺ T-cell responses (change < 10%) [2]

1. Human PAR2 membrane receptor competitive binding assay with Protease-Activated Receptor-2, amide (SLIGKV-NH₂): Purified membrane preparations from CHO cells stably expressing human PAR2 were incubated with a fixed concentration of [³H]2-furoyl-LIGRL-NH₂ (a selective PAR2 radioligand) and serial dilutions of Protease-Activated Receptor-2, amide (SLIGKV-NH₂) (10 nM–100 μM) in a buffer system (pH 7.4) containing Mg²⁺ and protease inhibitors. The mixture was incubated at 25℃ for 60 min to reach binding equilibrium, then filtered through glass fiber filters to separate receptor-bound and free radioligand. The radioactivity of the filters was quantified using a liquid scintillation counter, and non-specific binding was determined in the presence of excess unlabeled 2-furoyl-LIGRL-NH₂. The Ki value for competitive displacement was calculated using the Cheng-Prusoff equation based on the IC50 of radioligand displacement and the radioligand’s Kd (0.18 nM for [³H]2-furoyl-LIGRL-NH₂) [1]
2. PAR2-mediated calcium mobilization functional assay: CHO cells stably expressing human PAR2 were seeded in 96-well plates and loaded with a calcium-sensitive fluorescent dye for 30 min at 37℃. Protease-Activated Receptor-2, amide (SLIGKV-NH₂) (10 nM–10 μM) was added to the cells, and real-time changes in fluorescent intensity (excitation 485 nm, emission 520 nm) were monitored for 5 min using a microplate reader. The EC50 for calcium flux induction was derived by fitting the dose-response curve of fluorescent signal elevation, with maximal response normalized to that of the positive control (2-furoyl-LIGRL-NH₂) [1]

1. Murine BMDC PAR2 signaling and antigen uptake assay: Bone marrow cells were isolated from C57BL/6 mice femurs and tibias, cultured in medium supplemented with GM-CSF and IL-4 for 7 days to differentiate into BMDCs. The cells were then treated with serial concentrations of Protease-Activated Receptor-2, amide (SLIGKV-NH₂) (100 nM–5 μM) for 15 min (for MAPK phosphorylation) or 2 h (for antigen uptake). For MAPK analysis, cell lysates were prepared, and phosphorylated ERK1/2 and p38 MAPK levels were detected via western blot (normalized to total MAPK expression). For antigen uptake, FITC-labeled dextran was added to cells during compound treatment, and intracellular fluorescence intensity was measured via flow cytometry to quantify dextran internalization [2]
2. DC-T cell coculture proliferation and cytokine secretion assay: BMDCs were pretreated with Protease-Activated Receptor-2, amide (SLIGKV-NH₂) (1 μM) for 24 h and pulsed with OVA peptide (OVA323-339) for 4 h. Purified naive CD4⁺ T cells from OT-II transgenic mice (OVA-specific T-cell receptor) were labeled with a cell proliferation dye and cocultured with pretreated BMDCs (DC:T cell ratio = 1:10) for 72 h. T-cell proliferation was analyzed via flow cytometry (dye dilution), and culture supernatants were collected to measure IFN-γ concentration using a sandwich ELISA kit (absorbance detected at 450 nm) [2]

1. Murine DC antigen transport and T-cell activation assay with Protease-Activated Receptor-2, amide (SLIGKV-NH₂): Female C57BL/6 mice (6–8 weeks old, 18–22 g) were randomly divided into 2 groups (vehicle control, 500 μg/kg Protease-Activated Receptor-2, amide (SLIGKV-NH₂)), with 8 mice per group. The compound was dissolved in sterile PBS to prepare an injection solution (final concentration 5 mg/mL). The solution was administered via subcutaneous injection at a volume of 100 μL per mouse at the same site as subcutaneous OVA antigen (100 μg per mouse) injection. Vehicle control mice received the same volume of sterile PBS without the compound. At 24 h and 48 h post-administration, mice were euthanized, and DLNs were harvested to isolate single-cell suspensions. The number of OVA-loaded DCs was quantified via flow cytometry (gated on CD11c⁺MHC II⁺OVA⁺ cells), and OVA-specific T-cell proliferation and cytokine production were analyzed via flow cytometry (intracellular IFN-γ staining) and [³H]thymidine incorporation assay [2]

[1]. Binding of a highly potent protease-activated receptor-2 (PAR2) activating peptide, [3H]2-furoyl-LIGRL-NH2, to human PAR2. Br J Pharmacol. 2005 May;145(2):255-63.

[2]. Protease-activated receptor 2 signalling promotes dendritic cell antigen transport and T-cellactivation in vivo. Immunology. 2010 Jan;129(1):20-7.

Ser-Leu-Ile-Gly-Lys-Val-Amide is an oligopeptide.

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1. Protein-activated receptor-2 amide (SLIGKV-NH₂) is a synthetic PAR2 agonist peptide derived from the PAR2 line chain ligand sequence exposed after protease-mediated receptor cleavage[1][2]
2. Mechanism of action: This compound activates PAR2 by binding to the extracellular domain of the receptor (mimicking the natural line chain ligand), thereby triggering a G protein-dependent signaling cascade (including the MAPK pathway) and regulating cellular functions such as DC antigen uptake, migration, and T cell initiation[2]
3. Research applications: It is widely used as a selective PAR2 agonist in in vitro and in vivo studies to investigate PAR2-mediated immune responses, particularly DC-T cell communication in adaptive immunity. Immunological studies, and validation of PAR2 binding assays (as a comparison of high-affinity agonists such as [³H]2-furanoyl-LIGRL-NH₂) [1][2]
4. Receptor selectivity: This peptide is highly specific for PAR2 and does not cross-activate other PAR family members (PAR1/PAR3/PAR4) at pharmacologically relevant concentrations, making it a reliable tool for elucidating the specific biological functions of PAR2 [1]

C₂₈H₅₄N₈O₇

614.78

614.412

190383-13-2

10483914

Ser-Leu-Ile-Gly-Lys-Val-NH2

White to off-white solid powder

1.779

9

9

21

43

931

6

CC[C@@H]([C@H](NC([C@@H](NC([C@@H](N)CO)=O)CC(C)C)=O)C(NCC(N[C@H](C(N[C@H](C(N)=O)C(C)C)=O)CCCCN)=O)=O)C

HOWDUIVVWDUEED-WAUHAFJUSA-N

InChI=1S/C28H54N8O7/c1-7-17(6)23(36-27(42)20(12-15(2)3)34-25(40)18(30)14-37)28(43)32-13-21(38)33-19(10-8-9-11-29)26(41)35-22(16(4)5)24(31)39/h15-20,22-23,37H,7-14,29-30H2,1-6H3,(H2,31,39)(H,32,43)(H,33,38)(H,34,40)(H,35,41)(H,36,42)/t17-,18-,19-,20-,22-,23-/m0/s1

(2S)-6-amino-2-[[2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-2-amino-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]-3-methylpentanoyl]amino]acetyl]amino]-N-[(2S)-1-amino-3-methyl-1-oxobutan-2-yl]hexanamide

Ser-Leu-Ile-Gly-Lys-Val; PAR2 activating peptide; PAR2-AP; PAR-2 (1-6) Human; Protease-Activated Receptor-2, amide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ~33.3 mg/mL (~54.2 mM)

Solubility in Formulation 1: 100 mg/mL (162.66 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)