Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line

• DOI: 10.1084/jem.194.1.79
• Publication Date: 2002-07-26T16:48:33Z
• Authors: Andra B. Schromm; Egil Lien; Philipp Henneke; Jesse C. Chow; Atsutoshi Yoshimura; Holger Heine; Eicke Latz; Brian G. Monks; David A. Schwartz; Kensuke Miyake; Douglas T. Golenbock
• Keywords: Lipopolysaccharides; 0301 basic medicine; Membrane Glycoproteins; Interleukin-6; DNA Mutational Analysis; Genetic Complementation Test; Lipopolysaccharide Receptors; Lymphocyte Antigen 96; Receptors, Cell Surface; CHO Cells; Cell Line; Clone Cells; Endotoxins; 03 medical and health sciences; Cricetinae; Antigens, Surface; Mutation; Animals; Drosophila Proteins; Humans; Cloning, Molecular; Interleukin-1

Somatic cell mutagenesis is a powerful tool for characterizing receptor systems. We reported previously two complementation groups of mutant cell lines derived from CD14-transfected Chinese hamster ovary–K1 fibroblasts defective in responses to bacterial endotoxin. Both classes of mutants expressed a normal gene product for Toll-like receptor (TLR)4, and fully responded to stimulation by tumor necrosis factor (TNF)-α or interleukin (IL)-1β. We identified the lesion in one of the complementation groups in the gene for MD-2, a putative TLR4 coreceptor. The nonresponder phenotype of this mutant was reversed by transfection with MD-2. Cloning of MD-2 from the nonresponder cell line revealed a point mutation in a highly conserved region resulting in a C95Y amino acid exchange. Both forms of MD-2 colocalized with TLR4 on the cell surface after transfection, but only the wild-type cDNA reverted the lipopolysaccharide (LPS) nonresponder phenotype. Furthermore, soluble MD-2, but not soluble MD-2C95Y, functioned to enable LPS responses in cells that expressed TLR4. Thus, MD-2 is a required component of the LPS signaling complex and can function as a soluble receptor for cells that do not otherwise express it. We hypothesize that MD-2 conformationally affects the extracellular domain of TLR4, perhaps resulting in a change in affinity for LPS or functioning as a portion of the true ligand for TLR4.