The Journal of Experimental Medicine
IN Cell Analyzer 2000
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Published online 2 July 2001. doi:10.1084/jem.194.1.79
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© The Rockefeller University Press, 0022-1007/2001/7/79/ $5.00
The Journal of Experimental Medicine, Volume 194, Number 1, July 2, 2001 79-88

Original Article

Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line: A Point Mutation in a Conserved Region of Md-2 Abolishes Endotoxin-Induced Signaling



Andra B. Schromma,b, Egil Lienc, Philipp Hennekea, Jesse C. Chowd, Atsutoshi Yoshimurae, Holger Heineb, Eicke Latza, Brian G. Monksa, David A. Schwartzf, Kensuke Miyakeg, and Douglas T. Golenbocka

a Evans Biomedical Research Center, Boston University School of Medicine, Boston, MA 02118
b Research Center Borstel, 23845 Borstel, Germany
c Institute of Cancer Research and Molecular Biology, Norwegian University of Science and Technology, 7489 Trondheim, Norway
d Eisai Research Institute, Andover, MA 01810
e Nagasaki University School of Dentistry, Nagasaki 852-8588, Japan
f Duke University Medical Center, Durham, NC 27710
g Saga Medical School, Saga 849-8501, Japan
Rm. 615, Evans Biomedical Research Center, Boston University School of Medicine, 650 Albany St., Boston, MA 02118.617-414-5280617-414-7965

douglas.golenbock{at}bmc.org

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)-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.

Key Words: sepsis • signal transduction • Toll-like receptors • Gram-negative bacteria • lipopolysaccharide

Abbreviations used in this paper: bp, basepair; CHO, Chinese hamster ovary–K1 fibroblast; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, c-Jun NH2-terminal kinase; MAP, mitogen-activated protein; NF, nuclear factor; TLR, Toll-like receptor.

© 2001 The Rockefeller University Press


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