title: The ectodomain of the Toll-like receptor 4 prevents constitutive receptor activation.
abstract: Toll-like receptor 4 (TLR4) is involved in activation of the innate immune response in a large number of different diseases. Despite numerous studies, the role of separate domains of TLR4 in the regulation of receptor activation is poorly understood. Replacement of the TLR4 ectodomain with LPS-binding proteins MD-2 or CD14 resulted in a robust ligand-independent constitutive activation comparable with the maximal stimulation of the receptor with LPS. The same effect was achieved by the replacement of the ectodomain with a monomeric fluorescent protein or a 24-kDa gyrase B fragment. This demonstrates an intrinsic dimerization propensity of the transmembrane and cytoplasmic domains of TLR4 and reveals a previously unknown function of the ectodomain in inhibiting spontaneous receptor dimerization. Constitutive activation was abolished by the replacement of the ectodomain by a bulkier protein ovalbumin. N-terminal deletion variants of TLR4 revealed that the smallest segment of the ectodomain that already prevents constitutive activity comprises only 90 residues (542 to 631) of the total 608 residues. We conclude that TLR4 represents a receptor with a low threshold of activation that can be rapidly activated by the release of inhibition exerted by its ectodomain. This is important for the sensitivity of TLR4 to activation by different agonists. The TLR4 ectodomain has multiple roles in enabling ligand regulated activation, providing proper localization while serving as an inhibitor to prevent spontaneous, ligand-independent dimerization.
authors.affiliation: Department of Biotechnology, National Institute of Chemistry, University of Ljubljana, 1000 Ljubljana, Slovenia.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Panter
authors.forename: Roman
authors.initials: R
authors.lastname: Jerala
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000819:agonists / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D051218:Lymphocyte Antigen 96; D011993:Recombinant Fusion Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4