Expression of functional diphtheria toxin receptors on highly toxin-sensitive mouse cells that specifically bind radioiodinated toxin.
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.
Naglich
J G
JG
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235-9048.
Rolf
J M
JM
Eidels
L
L
D000818:Animals; D015139:Blotting, Southern; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004167:Diphtheria Toxin / Q000378:metabolism* / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D015723:Gene Library; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D007700:Kinetics; D007739:L Cells; D051379:Mice; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014709:Vero Cells
D004167:Diphtheria Toxin; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D004247:DNA