Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line
10.1084/jem.194.1.79
Crossref journal-article
Rockefeller University Press
The Journal of Experimental Medicine (291)
Abstract

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.

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Schromm, A. B., Lien, E., Henneke, P., Chow, J. C., Yoshimura, A., Heine, H., Latz, E., Monks, B. G., Schwartz, D. A., Miyake, K., & Golenbock, D. T. (2001). Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line. The Journal of Experimental Medicine, 194(1), 79–88.

Authors 11
  1. Andra B. Schromm (first)
  2. Egil Lien (additional)
  3. Philipp Henneke (additional)
  4. Jesse C. Chow (additional)
  5. Atsutoshi Yoshimura (additional)
  6. Holger Heine (additional)
  7. Eicke Latz (additional)
  8. Brian G. Monks (additional)
  9. David A. Schwartz (additional)
  10. Kensuke Miyake (additional)
  11. Douglas T. Golenbock (additional)
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Dates
Type When
Created 23 years, 1 month ago (July 26, 2002, 12:48 p.m.)
Deposited 2 years, 1 month ago (July 25, 2023, 6:58 a.m.)
Indexed 1 month, 3 weeks ago (July 2, 2025, 10:38 a.m.)
Issued 24 years, 1 month ago (July 2, 2001)
Published 24 years, 1 month ago (July 2, 2001)
Published Online 24 years, 1 month ago (July 2, 2001)
Published Print 24 years, 1 month ago (July 2, 2001)
Funders 0

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@article{Schromm_2001, title={Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line}, volume={194}, ISSN={1540-9538}, url={http://dx.doi.org/10.1084/jem.194.1.79}, DOI={10.1084/jem.194.1.79}, number={1}, journal={The Journal of Experimental Medicine}, publisher={Rockefeller University Press}, author={Schromm, Andra B. and Lien, Egil and Henneke, Philipp and Chow, Jesse C. and Yoshimura, Atsutoshi and Heine, Holger and Latz, Eicke and Monks, Brian G. and Schwartz, David A. and Miyake, Kensuke and Golenbock, Douglas T.}, year={2001}, month=jul, pages={79–88} }