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Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin.
J Biol Chem 2015; 290(6):3529-41JB

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+Show Affiliations

From the Department of Physiology, Center for Vascular and Inflammatory Diseases, and.From the Department of Physiology, Center for Vascular and Inflammatory Diseases, and.From the Department of Physiology, Center for Vascular and Inflammatory Diseases, and.From the Department of Physiology, Center for Vascular and Inflammatory Diseases, and.From the Department of Physiology, Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland 21201.From the Department of Physiology, Center for Vascular and Inflammatory Diseases, and tantalis@som.umaryland.edu.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

25519908

Citation

Driesbaugh, Kathryn H., et al. "Proteolytic Activation of the Protease-activated Receptor (PAR)-2 By the Glycosylphosphatidylinositol-anchored Serine Protease Testisin." The Journal of Biological Chemistry, vol. 290, no. 6, 2015, pp. 3529-41.
Driesbaugh KH, Buzza MS, Martin EW, et al. Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin. J Biol Chem. 2015;290(6):3529-41.
Driesbaugh, K. H., Buzza, M. S., Martin, E. W., Conway, G. D., Kao, J. P., & Antalis, T. M. (2015). Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin. The Journal of Biological Chemistry, 290(6), pp. 3529-41. doi:10.1074/jbc.M114.628560.
Driesbaugh KH, et al. Proteolytic Activation of the Protease-activated Receptor (PAR)-2 By the Glycosylphosphatidylinositol-anchored Serine Protease Testisin. J Biol Chem. 2015 Feb 6;290(6):3529-41. PubMed PMID: 25519908.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin. AU - Driesbaugh,Kathryn H, AU - Buzza,Marguerite S, AU - Martin,Erik W, AU - Conway,Gregory D, AU - Kao,Joseph P Y, AU - Antalis,Toni M, Y1 - 2014/12/17/ PY - 2014/12/19/entrez PY - 2014/12/19/pubmed PY - 2015/5/6/medline KW - Cell Surface KW - G-protein-coupled Receptor (GPCR) KW - PAR-2 KW - Protease KW - Protease-activated Receptor-2 KW - Serine Protease KW - Signaling KW - Testisin SP - 3529 EP - 41 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 290 IS - 6 N2 - 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. SN - 1083-351X UR - https://www.unboundmedicine.com/medline/citation/25519908/Proteolytic_activation_of_the_protease_activated_receptor__PAR__2_by_the_glycosylphosphatidylinositol_anchored_serine_protease_testisin_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=25519908 DB - PRIME DP - Unbound Medicine ER -