Description:
Abstract Toll-like receptor (TLR)4 signaling involves assembly of multi-protein complexes in lipid rafts. We identified Leucine rich repeats and calponin homology containing protein (Lrch)4 in a proteomic screen as a protein recruited to rafts of lipopolysaccharide (LPS)-exposed macrophages. Lrch4 had no published function, but structure predictions, including a transmembrane domain and leucine rich repeats, were suggestive of a receptor. Subcellular fractionation studies verified Lrch4 localization to the plasma membrane. qRT-PCR of 14 mouse tissues revealed that Lrch4 is ubiquitous. Lrch4 silencing in RAW 264.7 cells using lentiviral shRNAs and siRNAs attenuated LPS induction of TNFα and activation of mitogen-activated protein kinases and NF-κB. Lrch4 knockdown also reduced TNFα induction by ligands to several other TLRs. Lrch4 knockdown in TLR2 or TLR4 stable MD2-CD14-293 cells revealed that Lrch4 regulates human TLR but not TNFα -receptor signaling. Lrch4 and MD2 co-precipitated with biotin-LPS, and Lrch4 knockdown reduced cell binding of biotin-LPS without altering surface display of TLR4, together suggesting that Lrch4 interacts either directly or indirectly with LPS as part of a receptor complex. Lrch4 moreover co-precipitated with both MyD88 and TLR2. Lentiviral shRNA-mediated knockdown of Lrch4 in the murine airway reduced LPS-induced neutrophil influx into the airspaces in vivo. Taken together, we hypothesize that Lrch4 is a novel regulator of the innate immune response.