title: Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin.
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.
authors.affiliation: From the Department of Physiology, Center for Vascular and Inflammatory Diseases, and.
authors.forename: Kathryn H
authors.initials: KH
authors.lastname: Driesbaugh
authors.affiliation: From the Department of Physiology, Center for Vascular and Inflammatory Diseases, and.
authors.forename: Marguerite S
authors.initials: MS
authors.lastname: Buzza
authors.affiliation: From the Department of Physiology, Center for Vascular and Inflammatory Diseases, and.
authors.forename: Erik W
authors.initials: EW
authors.lastname: Martin
authors.affiliation: From the Department of Physiology, Center for Vascular and Inflammatory Diseases, and.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Conway
authors.affiliation: From the Department of Physiology, Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland 21201.
authors.forename: Joseph P Y
authors.initials: JP
authors.lastname: Kao
authors.affiliation: From the Department of Physiology, Center for Vascular and Inflammatory Diseases, and tantalis@som.umaryland.edu.
authors.forename: Toni M
authors.initials: TM
authors.lastname: Antalis
mesh_terms: D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism; D058851:GPI-Linked Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System; D016328:NF-kappa B / Q000378:metabolism; D059748:Proteolysis; D044464:Receptor, PAR-2 / Q000737:chemistry / Q000378:metabolism*; D020218:Response Elements; D012697:Serine Endopeptidases / Q000378:metabolism*
chemical_list: D058851:GPI-Linked Proteins; D015850:Interleukin-6; D016209:Interleukin-8; D016328:NF-kappa B; D044464:Receptor, PAR-2; C456340:Prss21 protein, mouse; D012697:Serine Endopeptidases
keywords: Cell Surface; G-protein-coupled Receptor (GPCR); PAR-2; Protease; Protease-activated Receptor-2; Serine Protease; Signaling; Testisin