title: Analysis of proteinase-activated receptor 2 and TLR4 signal transduction: a novel paradigm for receptor cooperativity.
abstract: 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-kappaB-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-kappaB reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple Gi 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-kappaB 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-kappaB 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 PAR2(-/-) 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.
authors.affiliation: Department of Microbiology and Immunology, University of Maryland, Baltimore, Maryland 21201, USA.
authors.forename: Prasad
authors.initials: P
authors.lastname: Rallabhandi
authors.forename: Quan M
authors.initials: QM
authors.lastname: Nhu
authors.forename: Vladimir Y
authors.initials: VY
authors.lastname: Toshchakov
authors.forename: Wenji
authors.initials: W
authors.lastname: Piao
authors.forename: Andrei E
authors.initials: AE
authors.lastname: Medvedev
authors.forename: Morley D
authors.initials: MD
authors.lastname: Hollenberg
authors.forename: Alessio
authors.initials: A
authors.lastname: Fasano
authors.forename: Stefanie N
authors.initials: SN
authors.lastname: Vogel
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000276:immunology; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000276:immunology; D000818:Animals; D002460:Cell Line; D016207:Cytokines / Q000235:genetics / Q000276:immunology; D005799:Genes, Dominant / Q000235:genetics / Q000276:immunology; D006801:Humans; D007113:Immunity, Innate / Q000187:drug effects / Q000235:genetics; D007249:Inflammation / Q000235:genetics / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000276:immunology; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology; D009842:Oligopeptides / Q000276:immunology / Q000494:pharmacology; D044464:Receptor, PAR-2 / Q000819:agonists / Q000235:genetics / Q000276:immunology*; D018123:Receptors, Interleukin / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000235:genetics / Q000276:immunology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D016207:Cytokines; D008070:Lipopolysaccharides; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D009842:Oligopeptides; D044464:Receptor, PAR-2; D018123:Receptors, Interleukin; D051197:Toll-Like Receptor 4; C507779:MYD88 protein, human; C507780:Myd88 protein, mouse; C476286:Ticam1 protein, mouse; C470409:TICAM1 protein, human; C480228:TICAM2 protein, human; C495345:TLR4 protein, human; C476416:Ticam2 protein, mouse; C493487:Tlr4 protein, mouse