Proteinase-activated receptors (PARs) -: the PAR3 neo-N-terminal peptide TFRGAP interacts with PAR1

Thrombin activates protemase-activated receptor (PAR)(1), PAR(3) and PAR(4) by a unique mechanism that involves cleavage of the receptor and exposure of a new N-terminal domain acting as a tethered ligand. Synthetic peptides based on the proteolytically revealed receptor sequence can selectively activate PAR(1) or PAR(4) independently of receptor cleavage. However, corresponding peptides for PAR(3) have not been identified thus far. Here, we demonstrate that the synthetic peptide TFRGAP representing the 1st six residues of the new amino terminus of PAR(3) induced ERK activation in human A-498 carcinoma cells endogeneously expressing PAR(1)(-/-) and PAR(3)(+). This effect was completely abolished by single alanine substitution at positions 3, 4 and 6 in the peptide. Since the specific PAR(1) antagonist RWJ 56110 completely abolished TFRGAP-induced ERK activation in A498 cells we speculate that TFRGAP does signal MARK via interaction with PAR(1). This was underlined by experiments on PAR(1)(-/-) mouse lung fibroblasts (KOLF cells) that stably overexpress human PAR(1) and PAR(3), respectively. While TFRGAP was without effect on ERK activation in PAR(3)(+) KOLF cells, it induced MARK activation in KOLF cells transfected with PAR(1). These studies provide evidence that analogues of the PAR(3) tethered ligand can mediate cell signaling by interaction with PAR(1)-type thrombin receptors. (C) 2004 Elsevier B.V. All rights reserved.