Toll-like receptors (TLRs) are sensors for the detection of invading infectious agents and can initiate innate immune responses. Because the innate immune system induces an appropriate defense against different pathogens, different TLR signaling domains may have unique properties that are responsible for eliciting distinctive responses to different types of pathogens. To test this hypothesis, we created ligand-regulated TLR chimeric receptors composed of the extracellular region of TLR4 and the transmembrane and cytoplasmic regions of other TLRs and expressed these chimeras in macrophages lacking endogenous TLR4. Interestingly, the chimeras between TLR4 and either TLR3, TLR7, or TLR9 were localized completely intracellularly whereas other chimeras were expressed on the cell surface. Lipopolysaccharide (LPS), a ligand for these chimeras, induced the activation of nuclear factor B and mitogenactivated protein kinases and the subsequent production of pro-inflammatory cytokines in macrophages expressing TLR4, TLR4/TLR5, or TLR4/TLR8 chimeras but not in macrophages expressing TLR4/TLR1, TLR4/TLR2, or TLR4/TLR6 chimeras. Co-expression of unresponsive chimeras in some combinations (chimeras with TLR1؉TLR2 or TLR2؉TLR6 but not TLR1؉TLR6) resulted in LPS responsiveness, indicating functional complementarity. Furthermore, the pair of TLR2؉TLR6 chimera required approximately 10-fold less LPS to induce the same responses compared with the TLR1؉TLR2 pair. Finally, LPS induced effective interferon-␤ production and subsequent Stat1 phosphorylation in macrophages expressing full-length TLR4 but not other cell surface TLR chimeras. These results suggest that the functions of TLRs are diversified not only in their extracellular regions for ligand recognition but also in their transmembrane and cytoplasmic regions for subcellular localization and signaling properties.

Toll-like receptor (TLR) 3 and TLR7 are indispensable for host defense against viral infection by recognizing virus-derived RNAs and are localized to intracellular membranes via an unknown mechanism. We recently reported experiments with chimeric Toll-like receptors that suggested that the subcellular distribution of TLRs may be defined by their transmembrane and/or cytoplasmic domains. Here we demonstrate that the intracellular localization of TLR3 is achieved by a 23-amino acid sequence (Glu 727 to Asp 749 ) present in the linker region between the transmembrane domain and Toll-interleukin 1 receptor resistance (TIR) domain. In contrast, the intracellular localization of TLR7 is achieved by its transmembrane domain. These elements also targeted a heterologous type I transmembrane protein CD25 to the intracellular compartment that contained TLR3 and TLR7. Despite their using distinct regulatory elements for intracellular localization, TLR3 was found to co-localize with TLR7. In addition, TLR3 and TLR7 were preferentially localized near phagosomes containing apoptotic cell particles. These findings reveal that TLR3 and TLR7 contain unique targeting sequences, which differentially lead them to the same intracellular compartments and adjacent to phagosomes containing apoptotic cell particles, where these receptors may access their ligands for the induction of immune responses against viral infection. Toll-like receptors (TLRs)2 are pattern-recognition receptors that detect highly conserved molecular structures of microorganisms or viruses and regulate both innate and adaptive immune responses against pathogens (1). More than 10 TLRs have been identified in human and mouse (2). TLR1, TLR2, TLR4, TLR5, and TLR6 recognize bacterial cell wall and cell surface components, such as lipoproteins, lipopolysaccharide, and flagellin. On the other hand, TLR3, TLR7, TLR8, and TLR9 recognize pathogen nucleic acids, such as viral RNAs and bacterial DNA (2, 3). All TLRs have a cytoplasmic signaling domain called the Toll/interleukin 1 receptor resistance (TIR) domain, which associates with intracellular TIR domain-containing adaptors, such as MyD88, TIRAP, TRIF/TICAM1, and TRAM/TICAM2. These TLR-associated adaptor molecules in turn mediate downstream signaling to induce pro-inflammatory and/or anti-viral innate immune responses (3).Since all TLRs are typical type I transmembrane proteins composed of an NH 2 -terminal signal peptide, an extracellular domain involved in ligand recognition, a single transmembrane domain, and a cytoplasmic domain, it was initially assumed that all TLRs would be expressed on the cell surface. However, studies using chimeric receptor approaches, fluorescently labeled TLRs, and anti-TLR antibodies have indicated that whereas TLR1, TLR2, TLR4, TLR5, and TLR6 are expressed on the cell surface, TLR3, TLR7, and TLR9 are completely localized in intracellular acidic compartments (4 -9). Based on data with chimeric receptors, TLR8 appears to be localized primarily intracellularly but with a small fraction on t...

Nitric oxide (NO) physiologically regulates numerous cellular responses through S-nitrosylation of protein cysteine residues. We performed antibody-array screening in conjunction with biotin-switch assays to look for S-nitrosylated proteins. Using this combination of techniques, we found that phosphatase with sequence homology to tensin (PTEN) is selectively S-nitrosylated by low concentrations of NO at a specific cysteine residue (Cys-83). S-nitrosylation of PTEN (forming SNO-PTEN) inhibits enzymatic activity and consequently stimulates the downstream Akt cascade, indicating that Cys-83 is a critical site for redox regulation of PTEN function. In ischemic mouse brain, we observed SNO-PTEN in the core and penumbra regions but found SNO-Akt, which is known to inhibit Akt activity, only in the ischemic core. These findings suggest that low concentrations of NO, as found in the penumbra, preferentially S-nitrosylate PTEN, whereas higher concentrations of NO, known to exist in the ischemic core, also S-nitrosylate Akt. In the penumbra, inhibition of PTEN (but not Akt) activity by S-nitrosylation would be expected to contribute to cell survival by means of enhanced Akt signaling. In contrast, in the ischemic core, SNO-Akt formation would inhibit this neuroprotective pathway. In vitro model systems support this notion. Thus, we identify unique sites of PTEN and Akt regulation by means of S-nitrosylation, resulting in an "on-off" pattern of control of Akt signaling.apoptosis | ischemia | oxidation N itric oxide (NO) exerts pleiotropic cellular responses on proliferation, apoptosis, neurotransmission, and neurotoxicity in several types of cells by means of protein S-nitrosylation. This modification occurs by means of oxidative reaction between NO and cysteine (Cys) thiol in the presence of an electron acceptor (such as O 2 or a transition metal) or through transnitrosylation from S-nitrosothiol to another Cys thiol (1-3). Several methods have been published to detect S-nitrosylated proteins (SNO-Ps) by using antibodies, photolysis, and mercury affinity (4). In particular, the biotin-switch assay is a modified immunoblot developed by Jaffrey and Snyder that has been commonly used to detect endogenous SNO-Ps; this method has greatly advanced the field (5). Subsequently, other methods have been developed to detect SNO-Ps (6), but some of them involve samples treated with high concentrations of NO donor. In the presence of high concentrations of NO, however, it is possible that some Cys residues are artifactually S-nitrosylated.Antibody arrays have been used to profile protein expression levels with high sensitivity. Each spotted antibody can be validated for its ability to bind proteins in the assay. Samples hybridizing to each antibody on the array can be easily detected. Although a number of proteins have been identified as substrates for S-nitrosylation in the past several years (3-6), we hypothesized that many more candidates modified by physiological levels of NO might still remain to be identified. We therefore teste...