NDLI: Expression of functional diphtheria toxin receptors on highly toxin-sensitive mouse cells that specifically bind radioiodinated toxin.

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Expression of functional diphtheria toxin receptors on highly toxin-sensitive mouse cells that specifically bind radioiodinated toxin.

Content Provider	PubMed Central
Author	Naglich, J. G. Rolf, J. M. Eidels, L.
Abstract	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.
Starting Page	2170
File Format	PDF
ISSN	10916490
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	6
Volume Number	89
Language	English
Publisher Date	1992-03-15
Access Restriction	Open
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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