Dual effect of the novel peptide antagonist K-14585 on Proteinase-Activated Receptor-2 (PAR2)-mediated signalling

F.G. Goh, P.Y. Ng, M. Reilly, T. Kanke, R. Plevin

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Here we have examined the effects of the novel peptide antagonist N-[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-5-yl]aminocarbonyl}-glycinyl-L-lysinyl-L-phenylalanyl-N-benzhydrylamide (K-14585) on proteinase-activated receptor (PAR)2-mediated intracellular signalling events. Using NCTC2544 cells expressing PAR2, we assessed the effects of K-14585 on PAR2-mediated [3H] inositol phosphate accumulation, MAP kinase activation, p65 NFκB phosphorylation and DNA binding and IL-8 production. Pretreatment with K-14585 (5 µM) inhibited [3H] inositol phosphate levels stimulated by PAR2-activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) in PAR2-expressing NCTC2544 cells. K-14585 pretreatment did not influence PAR2-mediated extracellular regulated kinase activation but inhibited p38 MAP kinase phosphorylation. At a higher concentration (30 µM), K-14585 alone stimulated p38 MAP kinase activation. These effects were replicated in EAhy926 cells, endogenously expressing PAR2, but not in parental or PAR4-expressing NCTC2544 cells, suggesting these effects were PAR2-dependent. SLIGKV-mediated stimulation of p38 MAP kinase phosphorylation was substantially reduced by the Gq/11 inhibitor YM-254890, without affecting K-14585-mediated phosphorylation. Pretreatment with K-14585 inhibited PAR2-mediated p65 NFκB phosphorylation and NFκB-DNA binding. K-14585 (30 µM) alone stimulated comparable NFκB reporter activity to SLIGKV-OH. K-14585 inhibited SLIGKV-stimulated IL-8 production, but given alone increased IL-8. While SLIGKV-induced IL-8 formation was reduced by both SB203580 and YM-254890, the response to K-14585 was sensitive to SB203580 but not YM-254890. These data reveal that K-14585 has a duality of action functioning both as an antagonist and agonist due to either partial agonist actions or possible agonist-directed signalling. The data also suggest two modes of p38 MAP kinase activation emanating from PAR2, one Gq/11-dependent and the other Gq/11-independent.
LanguageEnglish
Pages1695-1704
Number of pages9
JournalBritish Journal of Pharmacology
Volume158
Issue number7
DOIs
Publication statusPublished - Dec 2009

Fingerprint

PAR-2 Receptor
seryl-leucyl-isoleucyl--glycyl-lysyl-valine
Peptides
p38 Mitogen-Activated Protein Kinases
Interleukin-8
Phosphorylation
Inositol Phosphates
K-14585
eIF-2 Kinase
DNA

Keywords

  • PAR2
  • Gq/11 signalling
  • p38 MAP kinase
  • K-14585 peptide antagonist
  • inositol phosphate

Cite this

@article{32578e36504446fbbd47aefc19b5ff1a,
title = "Dual effect of the novel peptide antagonist K-14585 on Proteinase-Activated Receptor-2 (PAR2)-mediated signalling",
abstract = "Here we have examined the effects of the novel peptide antagonist N-[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-5-yl]aminocarbonyl}-glycinyl-L-lysinyl-L-phenylalanyl-N-benzhydrylamide (K-14585) on proteinase-activated receptor (PAR)2-mediated intracellular signalling events. Using NCTC2544 cells expressing PAR2, we assessed the effects of K-14585 on PAR2-mediated [3H] inositol phosphate accumulation, MAP kinase activation, p65 NFκB phosphorylation and DNA binding and IL-8 production. Pretreatment with K-14585 (5 µM) inhibited [3H] inositol phosphate levels stimulated by PAR2-activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) in PAR2-expressing NCTC2544 cells. K-14585 pretreatment did not influence PAR2-mediated extracellular regulated kinase activation but inhibited p38 MAP kinase phosphorylation. At a higher concentration (30 µM), K-14585 alone stimulated p38 MAP kinase activation. These effects were replicated in EAhy926 cells, endogenously expressing PAR2, but not in parental or PAR4-expressing NCTC2544 cells, suggesting these effects were PAR2-dependent. SLIGKV-mediated stimulation of p38 MAP kinase phosphorylation was substantially reduced by the Gq/11 inhibitor YM-254890, without affecting K-14585-mediated phosphorylation. Pretreatment with K-14585 inhibited PAR2-mediated p65 NFκB phosphorylation and NFκB-DNA binding. K-14585 (30 µM) alone stimulated comparable NFκB reporter activity to SLIGKV-OH. K-14585 inhibited SLIGKV-stimulated IL-8 production, but given alone increased IL-8. While SLIGKV-induced IL-8 formation was reduced by both SB203580 and YM-254890, the response to K-14585 was sensitive to SB203580 but not YM-254890. These data reveal that K-14585 has a duality of action functioning both as an antagonist and agonist due to either partial agonist actions or possible agonist-directed signalling. The data also suggest two modes of p38 MAP kinase activation emanating from PAR2, one Gq/11-dependent and the other Gq/11-independent.",
keywords = "PAR2, Gq/11 signalling, p38 MAP kinase, K-14585 peptide antagonist, inositol phosphate",
author = "F.G. Goh and P.Y. Ng and M. Reilly and T. Kanke and R. Plevin",
year = "2009",
month = "12",
doi = "10.1111/j.1476-5381.2009.00415.x",
language = "English",
volume = "158",
pages = "1695--1704",
journal = "British Journal of Pharmacology",
issn = "0007-1188",
number = "7",

}

Dual effect of the novel peptide antagonist K-14585 on Proteinase-Activated Receptor-2 (PAR2)-mediated signalling. / Goh, F.G.; Ng, P.Y.; Reilly, M.; Kanke, T.; Plevin, R.

In: British Journal of Pharmacology, Vol. 158, No. 7, 12.2009, p. 1695-1704.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Dual effect of the novel peptide antagonist K-14585 on Proteinase-Activated Receptor-2 (PAR2)-mediated signalling

AU - Goh, F.G.

AU - Ng, P.Y.

AU - Reilly, M.

AU - Kanke, T.

AU - Plevin, R.

PY - 2009/12

Y1 - 2009/12

N2 - Here we have examined the effects of the novel peptide antagonist N-[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-5-yl]aminocarbonyl}-glycinyl-L-lysinyl-L-phenylalanyl-N-benzhydrylamide (K-14585) on proteinase-activated receptor (PAR)2-mediated intracellular signalling events. Using NCTC2544 cells expressing PAR2, we assessed the effects of K-14585 on PAR2-mediated [3H] inositol phosphate accumulation, MAP kinase activation, p65 NFκB phosphorylation and DNA binding and IL-8 production. Pretreatment with K-14585 (5 µM) inhibited [3H] inositol phosphate levels stimulated by PAR2-activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) in PAR2-expressing NCTC2544 cells. K-14585 pretreatment did not influence PAR2-mediated extracellular regulated kinase activation but inhibited p38 MAP kinase phosphorylation. At a higher concentration (30 µM), K-14585 alone stimulated p38 MAP kinase activation. These effects were replicated in EAhy926 cells, endogenously expressing PAR2, but not in parental or PAR4-expressing NCTC2544 cells, suggesting these effects were PAR2-dependent. SLIGKV-mediated stimulation of p38 MAP kinase phosphorylation was substantially reduced by the Gq/11 inhibitor YM-254890, without affecting K-14585-mediated phosphorylation. Pretreatment with K-14585 inhibited PAR2-mediated p65 NFκB phosphorylation and NFκB-DNA binding. K-14585 (30 µM) alone stimulated comparable NFκB reporter activity to SLIGKV-OH. K-14585 inhibited SLIGKV-stimulated IL-8 production, but given alone increased IL-8. While SLIGKV-induced IL-8 formation was reduced by both SB203580 and YM-254890, the response to K-14585 was sensitive to SB203580 but not YM-254890. These data reveal that K-14585 has a duality of action functioning both as an antagonist and agonist due to either partial agonist actions or possible agonist-directed signalling. The data also suggest two modes of p38 MAP kinase activation emanating from PAR2, one Gq/11-dependent and the other Gq/11-independent.

AB - Here we have examined the effects of the novel peptide antagonist N-[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-5-yl]aminocarbonyl}-glycinyl-L-lysinyl-L-phenylalanyl-N-benzhydrylamide (K-14585) on proteinase-activated receptor (PAR)2-mediated intracellular signalling events. Using NCTC2544 cells expressing PAR2, we assessed the effects of K-14585 on PAR2-mediated [3H] inositol phosphate accumulation, MAP kinase activation, p65 NFκB phosphorylation and DNA binding and IL-8 production. Pretreatment with K-14585 (5 µM) inhibited [3H] inositol phosphate levels stimulated by PAR2-activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) in PAR2-expressing NCTC2544 cells. K-14585 pretreatment did not influence PAR2-mediated extracellular regulated kinase activation but inhibited p38 MAP kinase phosphorylation. At a higher concentration (30 µM), K-14585 alone stimulated p38 MAP kinase activation. These effects were replicated in EAhy926 cells, endogenously expressing PAR2, but not in parental or PAR4-expressing NCTC2544 cells, suggesting these effects were PAR2-dependent. SLIGKV-mediated stimulation of p38 MAP kinase phosphorylation was substantially reduced by the Gq/11 inhibitor YM-254890, without affecting K-14585-mediated phosphorylation. Pretreatment with K-14585 inhibited PAR2-mediated p65 NFκB phosphorylation and NFκB-DNA binding. K-14585 (30 µM) alone stimulated comparable NFκB reporter activity to SLIGKV-OH. K-14585 inhibited SLIGKV-stimulated IL-8 production, but given alone increased IL-8. While SLIGKV-induced IL-8 formation was reduced by both SB203580 and YM-254890, the response to K-14585 was sensitive to SB203580 but not YM-254890. These data reveal that K-14585 has a duality of action functioning both as an antagonist and agonist due to either partial agonist actions or possible agonist-directed signalling. The data also suggest two modes of p38 MAP kinase activation emanating from PAR2, one Gq/11-dependent and the other Gq/11-independent.

KW - PAR2

KW - Gq/11 signalling

KW - p38 MAP kinase

KW - K-14585 peptide antagonist

KW - inositol phosphate

UR - http://dx.doi.org/10.1111/j.1476-5381.2009.00415.x

U2 - 10.1111/j.1476-5381.2009.00415.x

DO - 10.1111/j.1476-5381.2009.00415.x

M3 - Article

VL - 158

SP - 1695

EP - 1704

JO - British Journal of Pharmacology

T2 - British Journal of Pharmacology

JF - British Journal of Pharmacology

SN - 0007-1188

IS - 7

ER -