Dual effect of the novel peptide antagonist K-14585 on proteinase-activated receptor-2-mediated signalling.

Background And Purpose: 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.

Experimental Approach: Using NCTC2544 cells expressing PAR(2), we assessed the effects of K-14585 on PAR(2)-mediated [(3)H] inositol phosphate accumulation, MAP kinase activation, p65 NFkappaB phosphorylation and DNA binding and IL-8 production.

Key Results: Pretreatment with K-14585 (5 microM) inhibited [(3)H] inositol phosphate levels stimulated by PAR(2)-activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) in PAR(2)-expressing NCTC2544 cells.

G-protein-dependent and -independent pathways regulate proteinase-activated receptor-2 mediated p65 NFkappaB serine 536 phosphorylation in human keratinocytes.

The mechanisms underpinning the coupling of GPCRs, such as PAR-2, to the phosphorylation of p65 NFkappaB have not been investigated. In the current study we found that trypsin and the selective PAR-2 activating peptide, 2f-LIGKV-OH, stimulated large and sustained increases in the serine 536 phosphorylation of p65/RelA in a transfected skin epithelial cell line and primary keratinocytes. Parallel experiments showed that in both cell types, p65 NFkappaB phosphorylation is mediated through the selective activation of IKK2.