Ligand Cross-reactivity within the Protease-activated Receptor Family

Abstract

Recently, a second member of the protease-activated receptor (PAR) family, named PAR-2, has been identified. Similar to the thrombin receptor, PAR-2 appears to be activated by proteolytic-mediated exposure of a "tethered ligand" sequence and can also be activated by the corresponding synthetic peptides. Similarities in the amino acid sequence of the receptors' tethered ligand sequences suggest that their respective agonist peptides might not be absolutely specific for their particular receptors. To test this, the receptor specificity of each agonist has been determined by measuring the responses of Xenopus oocytes expressing the thrombin receptor or PAR-2 to agonist peptides or enzymes. Thrombin receptors responded to thrombin, the human thrombin receptor-activating peptide SFLLRNP-NH2 (TRAP) (EC50 = 0.1 microM), and Xenopus TRAP, TFRIFD-NH2 (EC50 = 1 microM), but did not show any increase in calcium efflux over control levels with trypsin (50 nM) or PAR-2 agonist peptides (100 microM). Human and murine PAR-2 receptors responded comparably to human and murine PAR-2 agonist peptides (SLIGKVD and SLIGRL, respectively) (EC50 = 0.5-2.0 microM) and trypsin, but not to thrombin. PAR-2 was also found to be responsive to TRAP (EC50 = 1 microM) but was unresponsive to Xenopus TRAP (50 microM). Responses to additional peptide agonist analogs suggest that an amino-terminal serine is critical for PAR-2 agonist activity.

Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2

“…This suggests that colonic cancer cells may have an exquisite sensitivity to trypsin. However, the effects of trypsin on colonic cancer cell proliferation are clearly mediated by PAR-2 since we showed that the specific agonist of PAR-2, AP2 (Blackhart et al, 1996;Hollenberg et al, 1997) also promoted cell proliferation, mimicking the trypsin effect. In contrast the reverse peptide with the reverse AP2 peptide sequence was devoid of any mitogenic effect.…”

Activity of recombinant trypsin isoforms on human proteinase‐activated receptors (PAR): mesotrypsin cannot activate epithelial PAR‐1, ‐2, but weakly activates brain PAR‐1

“…PAR-2 seems to be activated physiologically mainly by trypsin (Nystedt et al, 1995). PAR-1 and PAR-4 are also activated by trypsin, although these two PARs are considered predominantly as thrombin receptors (Vouret-Craviari et al, 1995;Blackhart et al, 1996;Xu et al, 1998). To date no attempts have been undertaken to explore whether the various isoforms of trypsin differ in their ability to activate PARs.…”

Protease activated receptors: theme and variations

“…Early studies of PAR1 used agonist peptides that were later shown to activate PAR2 (Blackhart et al, 1996;Hollenberg et al, 1997). Over time, relatively speci®c agonist peptides have been described for PAR1, PAR2, and PAR4 (Table 1) (Blackhart et al, 1996;Xu et al, 1998;Faruqi et al, 2000;Hollenberg et al, 1997;Brass et al, 1992). In some cells, one can now tease out contributions from individual PAR subtypes, and con®rm distribution data with functional correlates of receptor expression.…”

“…Granzyme A (Suidan et al, 1994(Suidan et al, , 1996. c Structure/activity relationships have been explored in some detail for short peptides (up to 14 amino acids) that activate PAR1 and PAR2 Scarborough et al, 1992;Al-Ani et al, 1999;Hollenberg et al, 1992Hollenberg et al, , 1997Blackhart et al, 1996;Chao et al, 1992;Natarajan et al, 1995;Kawabata et al, 1999), and more recently for PAR4 (Kahn et al, 1998b(Kahn et al, , 1999Hollenberg et al, 1999;Faruqi et al, 2000). To date, no peptide activators of PAR3 have been described (Hollenberg, 1999).…”