Recognition of Double-stranded RNA by Human Toll-like Receptor 3 and Downstream Receptor Signaling Requires Multimerization and an Acidic pH

Abstract

Studies involving Toll-like receptor 3 (TLR3)-deficient mice suggest that this receptor binds double-stranded RNA. In the present study, we analyzed ligand/receptor interactions and receptor-proximal events leading to TLR3 activation. The mutagenesis approach showed that certain cysteine residues and glycosylation in TLR3 amino-terminal leucine-rich repeats were necessary for ligand-induced signaling. Furthermore, inactive mutants had a dominant negative effect, suggesting that the signaling module is a multimer. We constructed a chimeric molecule fusing the amino-terminal ectodomain of TLR3 to the transmembrane and carboxyl terminal domains of CD32a containing an immunoreceptor tyrosine-based motif. Expression of TLR3-CD32 in HEK293T cells and the myeloid cell line U937 resulted in surface localization of the receptor, whereas the nonrecombinant molecule was intracellularly local- Mammalian Toll-like receptors (TLRs) 7 belong to a family of receptors that recognize pathogen-associated molecular patterns. TLRs play a key role in host defense during pathogen infection by regulating and linking the innate and adaptive immune responses (1-3). TLRs are expressed in dendritic cells (DC), sentinels of the immune system, endowing them with the capacity to sense pathogen-derived products and to alert the immune system (4). Members of the TLR family are also variably expressed on nonhematopoietic cells. TLR-deficient mice and transfected cell lines have been the keys to understanding TLR function. Ligand specificity has been elucidated for most TLRs; thus, TLR2 and TLR4 recognize Gram-positive and Gram-negative bacterial cell wall products, respectively. TLR5 recognizes a structural epitope of bacterial flagellin, and TLR7, TLR8, and TLR9 have been demonstrated to recognize different forms of microbial-derived nucleic acid (5). Host-derived ligands for the TLRs have also been identified; in particular, TLR4 recognizes heat shock proteins and pulmonary surfactant (6), and TLR9 recognizes chromatin-IgG complexes (7).TLR3 has been extensively characterized to be a receptor for poly(I-C), a synthetic double-stranded RNA (dsRNA) mimic (8); recently, it has been shown to mediate responses to West Nile virus (9) as well as dsRNA derived from the helminth parasite Schistosoma (10). Additionally, host-derived mRNA has recently been shown to activate TLR3, suggesting that activation via TLR3 can occur in a variety of situations (11). Whereas most TLRs recruit myeloid differentiation factor 88 (12), with some variation in the signaling profile mediated by the additional recruitment of Toll/interleukin-1 receptor-containing adapter protein and TRIF-related adaptor molecule, TLR3 recruits only TRIF (13-15). TRIF can activate both NF-B through TRAF6 and receptor-interacting protein-1 (16) and interferon-regulatory factor 3 through IB kinase/ TANK-binding kinase 1 (17) and phosphoinositide 3-kinase (18). Although the signaling pathway of TLRs is increasingly well characterized, the parameters controlling interactions between the recept...

TLR3 Ligation Activates an Antiviral Response in Human Fetal Astrocytes: A Role for Viperin/cig5

“…1A). Based on the recently elucidated crystal structure of TLR3 (18) and the fact that the receptor is largely expressed on intracellular vesicles (data not shown), it has been proposed that a low pH environment is required for the receptor to recognize, bind, and signal in response to ligand (19,20). Therefore, we next attempted to inhibit TLR-3 signaling in our cells using the base acridine orange.…”

Cell proliferation and survival induced by Toll-like receptors is antagonized by type I IFNs

“…Peripheral blood mononuclear cells were purified by FicollHypaque centrifugation. Monocyte-derived DC (MonoDCs) were prepared as described (13). For growth factor and Western blot studies, MonoDCs were arrested by washing three times with PBS and were then replenished with serum, GM-CSF, and IL-4-free medium 24 h before activation.…”

“…In certain cell types, TRIF-dependent TLR signaling results in apoptosis with a mechanism that, in part, depends on the production of type I IFN (6)(7)(8)(9). However, in addition to apoptosis, TLR signaling may also result in cellular proliferation (10)(11)(12)(13).…”

Proteolytic Processing Regulates Toll-like Receptor 3 Stability and Endosomal Localization

“…They serve as sites for the sequestration of non-self-nucleic acids. The affinity of ligand recognition by TLR3 and TLR9 increases with the acidic environment found in endosomes (5,9,10). Endosomes also contain pH-activated proteases such as cathepsins that have been documented to cleave TLR7 and TLR9 (11)(12)(13).…”