Abstract
Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca(2+) mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface.
Highlights
Protease-activated receptors (PARs)-2 is a protease-activated, G-protein-coupled receptor involved in inflammation, development, and immunity
The serine protease inhibitors AEBSF, leupeptin, and aprotinin significantly inhibited rTestisin proteolytic activity, whereas the metalloproteinase inhibitor EDTA had no effect on rTestisin activity (Fig. 1B)
We provide the first evidence for the activation of PAR-2 by a GPI-anchored serine protease
Summary
PAR-2 is a protease-activated, G-protein-coupled receptor involved in inflammation, development, and immunity. Human PAR-2 has been shown to mediate cellular responses in vitro following cleavage at Arg by several serine proteases, including trypsin, trypsin IV, tryptase, kallikrein 4, and Factors VIIa (FVIIa) and FXa. The FVIIa-FXa complex must be anchored to the cell surface bound to tissue factor (FVIIa-FXa-TF) to activate PAR-2 [4, 7]. The truncated recombinant catalytic domains of several of the type II transmembrane serine proteases have been shown to proteolytically activate PAR-2 in vitro, including matriptase, HAT, and TMPRSS2 [11,12,13,14,15] Whether this activity occurs while these proteases are anchored on the cell surface has been less well studied, with the exception of matriptase, which has been shown to activate PAR-2 when expressed as a full-length protein in cells (16 –19). We report that testisin functions as a cell surface activator of PAR-2, inducing PAR-2 N-terminal cleavage and internalization and the activation of several PAR-2-dependent intracellular signaling pathways
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