UNC93B1 Binding and Signaling Nucleotide-Sensing TLRs Are Important for Juxtamembrane Region of the Acidic Amino Acid Residues in the

TLRs are divided into two groups based on their subcellular localization patterns. TLR1, 2, 4, 5, and 6 are expressed on the cell surface, whereas the nucleotide-sensing TLRs, such as TLR3, 7, 8, and 9 stay mainly inside cells. The polytopic membrane protein UNC93B1 physically interacts with the nucleotide-sensing TLRs and delivers them from the endoplasmic reticulum to endolyso-somes, where the TLRs recognize their ligands and initiate signaling. In cells with nonfunctional UNC93B1, the nucleic acid–sensing TLRs fail to exit the endoplasmic reticulum and consequently do not signal. However, the detailed molecular mechanisms that underlie the UNC93B1-mediated TLR trafficking remain to be clarified. All nucleotide-sensing TLRs contain acidic amino acid residues in the juxtamembrane region between the leucine-rich repeat domain and the transmembrane segment. We show that the D812 and E813 residues of TLR9 and the D699 and E704 residues of TLR3 help to determine the interaction of these TLRs with UNC93B1. Mutation of the acidic residues in TLR3 and TLR9 prevents UNC93B1 binding, as well as impairs TLR trafficking and renders the mutant receptors incapable of transmitting signals. Therefore, the acidic residues in the juxtamembrane region of the nucleotide-sensing TLRs have important functional roles. F oreign DNA and RNA derived from invading viruses and bacteria are sensed by several families of innate immune receptors, including TLRs, RIG-I-like receptors, and PYHIN family proteins, all of which can activate inflammatory responses (1–3). Because endogenous nucleic acids can also activate these receptors in pathogenic contexts and facilitate in-flammatory or autoimmune diseases, the host must have evolved mechanisms to discriminate microbial nucleic acids from endog-enous ones (4–7). One strategy for such discrimination is se-questration of the nucleotide-sensing receptors inside cells (8, 9). Although capable of sensing DNA and RNA from viruses or phagocytosed bacteria that enter the cytoplasm or endosomes, the intracellular receptors can avoid encountering a high concentration of extracellular host nucleic acids that are released from damaged cells. Among TLRs, TLR3, 7, 8, 9, and 13 recognize RNA, DNA, or small nucleotide analogs (10–15). Unlike the other TLRs that are expressed on the plasma membrane, these nucleotide-sensing TLRs are largely found inside cells (16, 17). Mice engineered to artificially express TLR9 on the cell surface succumb to fatal systemic inflammation, underscoring the physiological importance of intracellular localization of the nucleotide-sensing receptors (18). TLR9 resides in the endoplasmic reticulum (ER) in resting cells and is recruited to endolysosomes upon stimulation with its …