Proteolytic activation of the protease-activated receptor (PAR)-2 by the GPI-anchored serine protease testisin [Cell Biology]

December 17th, 2014 by Driesbaugh, K. H., Buzza, M. S., Martin, E. W., Conway, G. D., Kao, J. P. Y., Antalis, T. M.

Protease-activated receptors (PARs) are a family of seven transmembrane, G-protein-coupled receptors which 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 calcium 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 intracellular serum response element (SRE) and NFkappaB 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.