Raftlin Controls Lipopolysaccharide-Induced TLR4 Internalization and TICAM-1 Signaling in a Cell Type-Specific Manner

The clathrin-dependent endocytic pathway is crucial for endosomal TLR3- and TLR4-mediated Toll-IL-1R domain-containing adaptor molecule-1 (TICAM-1) signaling. TLR4 uses a different signaling platform, plasma membrane and endosomes, for activation of TIRAP-MyD88 and TICAM-2-TICAM-1, respectively. LPS-induced endocytosis of TLR4 is mandatory for TICAM-1-mediated signaling including IFN-β production. Several molecules/mechanisms such as CD14, clathrin, and phosphatidylinositol metabolism have been reported to act as inducers of TLR4 translocation. However, the molecular mechanism of spatiotemporal regulation of TLR4 signaling remains unresolved. We have previously shown that Raftlin is essential for clathrin-dependent endocytosis of TLR3 ligand in human epithelial cells and myeloid dendritic cells (DCs). In this article, we demonstrate that Raftlin also mediated LPS-induced TLR4 internalization and TICAM-1 signaling in human monocyte-derived DCs and macrophages (Mo-Mϕs). When Raftlin was knocked down, LPS-induced TLR4-mediated IFN-β promoter activation, but not NF-κB activation, was decreased in HEK293 cells overexpressing TLR4/MD-2 or TLR4/MD-2/CD14. LPS-induced IFN-β production by monocyte-derived DCs and Mo-Mϕs was significantly decreased by knockdown of Raftlin. Upon LPS stimulation, Raftlin moved from the cytoplasm to the plasma membrane in Mo-Mϕs, where it colocalized with TLR4. Raftlin associated with clathrin-associated adaptor protein-2 in resting cells and transiently bound to TLR4 and clathrin at the cell surface in response to LPS. Thus, Raftlin appears to modulate cargo selection as an accessary protein of clathrin-associated adaptor protein-2 in clathrin-mediated endocytosis of TLR3/4 ligands.