Interactions of Mast Cell Tryptase with Thrombin Receptors and PAR-2

Marina Molino,Elliot S Barnathan,Robert Numerof,Jim Clark,Mark Dreyer,Albana Cumashi,James A Hoxie,Norman Schechter,Marilyn Woolkalis,Lawrence F Brass

doi:10.1074/jbc.272.7.4043

Abstract

Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-protein-coupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca2+. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca2+. These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.

Highlights

Tryptase is a serine protease secreted by mast cells that is able to activate other cells
One piece of evidence supporting this mechanism of activation is that synthetic peptides corresponding to the tethered ligand domains of the thrombin receptor and PAR-2 are able to act as full agonists on their respective receptors [12, 13]
PAR-2 was thought to be a potential substrate for tryptase, because an earlier analysis of the P3P2P1 site preferences for tryptase in cleaving tripeptide nitroanilide substrates had revealed that the highest catalytic efficiency was for the tripeptide, Lys-Gly-Arg (ϳ4 ϫ 105 MϪ1 sϪ1) [29], which matches the P3P2P1 sequence at the activating cleavage site of human PAR-2 (Fig. 1)

Summary

Much less is known about the proteases that can activate

PAR-2, trypsin is clearly among them and thrombin is not [13, 18]. Based upon the apparent tissue distribution of PAR-2 mRNA, it seems unlikely that trypsin is the sole protease capable of activating it, but others have not yet been described. Some cells, including platelets and several megakaryoblastic cell lines, express thrombin receptors (12, 19 –22) but not PAR-2 [23, 24]. When added to intact receptors, tryptase cleaved endogenous and transfected PAR-2 It cleaved thrombin receptors overexpressed in COS-1 cells but did not activate endogenous thrombin receptors on platelets or megakaryoblastic CHRF-288 cells. This failure to activate endogenous thrombin receptors appears not to be due to a cathepsin G-like cleavage of a secondary site within the receptor N terminus but may reflect the relatively slower rate of hydrolysis suggested by peptide studies. In addition to identifying the first protease other than trypsin that can activate PAR-2, these results suggest that factors in addition to receptor primary sequence limit the ability of proteases to evoke responses from this subfamily of Gprotein-coupled receptors

EXPERIMENTAL PROCEDURES

RESULTS

Trypsin Thrombin

Not cleaved

DISCUSSION