Toll-like receptors (TLRs) are trans-membrane proteins that recognize pathogen associated molecular patterns (PAMPs) and subsequently induce an intracellular signaling cascade that triggers the activation of transcription factors which initiate the expression of host defense proteins. Ten human, structurally highly conserved TLRs have been identified and assigned to structurally very dissimilar ligands. The complexity of a PAMP-TLR interface is best understood for the recognition of Lipopolysaccharide (LPS) by a LBP-Cd14,TLR4-Md2 receptor complex. The central hypothesis of this project is that many further ligand specific co-receptors and co-ligands remain to be discovered. I propose to systematically search for endosomal TLR receptor complexes required for the innate immune response to viral infection. Therefore, an interdisciplinary strategy combining molecular biology, cell biology, chemistry and proteomics will be employed. Recombinant ectodomains will be purified and used for monoclonal antibody production and affinity chromatography with macrophage lysates to fish cellular binding proteins. Following pre-enrichment of endosomes, endogenous TLRs will be immunoprecipitated from macrophages. Moreover, ligand affinity chromatography based proteomics using synthetic analogues like Poly(I:C) or the small compound imiquimod and tandem affinity purification of TLRs provide additional methods for complex purifications. Following identification of complexes by mass spectrometry, interactors will be validated and further characterized using cell biological, biochemical and immunological methods. This project will help to understand the process of viral pathogen recognition by the innate immune system and can provide novel drug targets for the tuning of innate immune response.