Molecular genetic analysis of an endotoxin nonresponder mutant cell line: a point mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling.
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)-alpha or interleukin (IL)-1 beta. 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-2(C95Y), 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.
Schromm
A B
AB
Evans Biomedical Research Center, Boston University School of Medicine, Boston, MA 02118, USA.
Lien
E
E
Henneke
P
P
Chow
J C
JC
Yoshimura
A
A
Heine
H
H
Latz
E
E
Monks
B G
BG
Schwartz
D A
DA
Miyake
K
K
Golenbock
D T
DT
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics* / Q000378:metabolism; D016466:CHO Cells / Q000187:drug effects; D002460:Cell Line; D002999:Clone Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004252:DNA Mutational Analysis; D029721:Drosophila Proteins; D004731:Endotoxins / Q000378:metabolism*; D005816:Genetic Complementation Test; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D015850:Interleukin-6 / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism / Q000633:toxicity; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000954:Antigens, Surface; D029721:Drosophila Proteins; D004731:Endotoxins; D007375:Interleukin-1; D015850:Interleukin-6; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha