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 (DT(S)) colony was isolated. The purified DT(S) mouse cells are highly toxin-sensitive; they are at least 1000-fold more sensitive than wild-type L-M cells and only almost-equal-to 10-fold less sensitive than Vero cells. Incubation of the DT(S) 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 DT(S) 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 M(r) 6000 carboxyl-terminal receptor-binding domain, inhibited the binding of radioiodinated toxin to these DT(S) mouse cells to the same extent as unlabeled DT. With these DT(S) mouse cells as a source of monkey cDNA, it should be possible to clone the gene encoding the DT receptor.