Document Detail

Expression of functional diphtheria toxin receptors on highly toxin-sensitive mouse cells that specifically bind radioiodinated toxin.
MedLine Citation:
PMID:  1549577     Owner:  NLM     Status:  MEDLINE    
Diphtheria toxin (DT), a bacterial protein exotoxin, inactivates mammalian cell elongation factor 2 after toxin internalization by receptor-mediated endocytosis. To isolate the DT receptor, we cotransfected DT-resistant wild-type mouse L-M cells with a cDNA library constructed from RNA of highly toxin-sensitive monkey Vero cells and with a neomycin-resistance gene. Stably transfected G418-resistant L-M colonies were screened for DT sensitivity in a replica plate assay. After screening of 8000 colonies, one DT-sensitive (DTS) colony was isolated. The purified DTS mouse cells are highly toxin-sensitive; they are at least 1000-fold more sensitive than wild-type L-M cells and only approximately 10-fold less sensitive than Vero cells. Incubation of the DTS mouse cells with CRM 197, a nontoxic form of DT that competitively inhibits the binding of native DT to the toxin receptor, protected them from DT-mediated toxicity. More important, these DTS mouse cells express receptors on their cell surface that bind radioiodinated DT in a specific fashion, a property hitherto readily demonstrable only with highly toxin-sensitive cells of monkey origin. Furthermore, HA6DT, a DT fragment comprising the Mr 6000 carboxyl-terminal receptor-binding domain, inhibited the binding of radioiodinated toxin to these DTS mouse cells to the same extent as unlabeled DT. With these DTS mouse cells as a source of monkey cDNA, it should be possible to clone the gene encoding the DT receptor.
J G Naglich; J M Rolf; L Eidels
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  89     ISSN:  0027-8424     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  1992 Mar 
Date Detail:
Created Date:  1992-04-17     Completed Date:  1992-04-17     Revised Date:  2010-09-07    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  2170-4     Citation Subset:  IM    
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235-9048.
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MeSH Terms
Blotting, Southern
Cell Survival / drug effects
DNA / genetics,  isolation & purification
Diphtheria Toxin / metabolism*,  pharmacology
Drug Resistance / genetics
Gene Library
Intercellular Signaling Peptides and Proteins
Iodine Radioisotopes
L Cells (Cell Line)
Receptors, Cell Surface*
Receptors, Cholinergic / genetics,  metabolism*
Vero Cells
Grant Support
Reg. No./Substance:
0/Diphtheria Toxin; 0/Intercellular Signaling Peptides and Proteins; 0/Iodine Radioisotopes; 0/Receptors, Cell Surface; 0/Receptors, Cholinergic; 149176-25-0/heparin-binding EGF-like growth factor; 9007-49-2/DNA

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