The effect of receptor rapid‐internalization signals on diphtheria toxin endocytosis and cell sensitivity

Diphtheria toxin enters toxin‐sensitive mammalian cells by receptor‐mediated endocytosis employing the heparin‐binding EGF‐like growth factor precursor as its receptor. We reported previously (Almond and Eidels, 1994) that cytoplasmic domain mutants of the toxin receptor and cells expressing wild‐type receptor internalize toxin slowly, the rate being approximately that of normal turnover of the plasma membrane. To determine whether it was possible to increase toxin sensitivity by increasing the rate of toxin internalization, we constructed diphtheria toxin cytoplasmic domain mutant cell lines containing rapid‐internalization signals from either the low density lipoprotein receptor or from the lysosomal acid phosphatase precursor. Although cells transfected with mutant receptor genes internalized toxin at a faster rate than those expressing the wild‐type receptor, they showed a decrease in toxin sensitivity. This decreased sensitivity may be accounted for by an observed decrease in the number of toxin‐binding sites and by an increased rate of toxin internalization and degradation. These results suggest that the rate of toxin internalization may not be the rate‐limiting step in the cytotoxic process.