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J. Biol. Chem., Vol. 283, Issue 36, 24314-24325, September 5, 2008

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Analysis of Proteinase-activated Receptor 2 and TLR4 Signal Transduction

A NOVEL PARADIGM FOR RECEPTOR COOPERATIVITY^*

Prasad Rallabhandi¹, Quan M. Nhu¹, Vladimir Y. Toshchakov, Wenji Piao, Andrei E. Medvedev, Morley D. Hollenberg, Alessio Fasano^¶, and Stefanie N. Vogel²

From the Department of Microbiology and Immunology and ^¶Department of Pediatrics and the Mucosal Biology Research Center, University of Maryland, Baltimore, Maryland 21201 and the Department of Pharmacology and Therapeutics and Department of Medicine, University of Calgary, Calgary, T2N4N1, Alberta, Canada

Proteinase-activated receptor 2 (PAR₂), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR₂, often measured by Ca²⁺ release. PAR₂ contributes to inflammation through activation of NF-B-regulated genes; however, the mechanism by which this occurs is unknown. Overexpression of human PAR₂ in HEK293T cells resulted in concentration-dependent, PAR₂ AP-inducible NF-B reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple G_i proteins or sequester intracellular Ca²⁺. Because previous studies described synergistic PAR₂- and TLR4-mediated cytokine production, we hypothesized that PAR₂ and TLR4 might interact at the level of signaling. In the absence of TLR4, PAR₂-induced NF-B activity was inhibited by dominant negative (DN)-TRIF or DN-TRAM constructs, but not by DN-MyD88, findings confirmed using cell-permeable, adapter-specific BB loop blocking peptides. Co-expression of TLR4/MD-2/CD14 with PAR₂ in HEK293T cells led to a synergistic increase in AP-induced NF-B signaling that was MyD88-dependent and required a functional TLR4, despite the fact that AP exhibited no TLR4 agonist activity. Co-immunoprecipitation of PAR₂ and TLR4 revealed a physical association that was AP-dependent. The response to AP or lipopolysaccharide was significantly diminished in TLR4^–/– and PAR ^–/–₂ macrophages, respectively, and SW620 colonic epithelial cells exhibited synergistic responses to co-stimulation with AP and lipopolysaccharide. Our data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.

Received for publication, June 24, 2008

^* This work was supported, in whole or in part, by National Institutes of Health Grants R37 AI-18797 (to S. N. V.), R01 AI-059524 (to A. E. M.), R01 DK048373 (to A. F.), and T32 AI-07540 (to Q. M. N.), and a Canadian Institutes of Health Research operating grant (to M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Maryland, 660 W. Redwood St., Rm. 324, Baltimore, MD 21201. Tel.: 410-706-4838; Fax: 410-706-8607; E-mail: svogel{at}som.umaryland.edu.

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