Glutamic acid 141 of the diphtheria toxin receptor (HB-EGF precursor) is critical for toxin binding and toxin sensitivity.

The transmembrane precursor of the monkey heparin-binding EGF-like growth factor also functions as a diphtheria toxin receptor. The mouse precursor does not bind the toxin. Previously, the most important region for binding the toxin in the monkey precursor was narrowed down to residues 122-148 through the expression of chimeric mouse/monkey precursors and subsequent toxin-sensitivity assays. To define further the toxin binding domain of the monkey precursor, distinct monkey/mouse chimeric precursors were expressed and assayed. The region between monkey residues 136-148 was found to be absolutely necessary for the retention of toxin sensitivity. Within this region, the monkey and mouse precursors differ in only two residues (residues 141 and 147). A toxin-insensitive monkey/mouse chimera that contained monkey residues 1-136 was converted to a toxin-sensitive chimera by the mutation of a single residue (His141 to Glu141). Expression of a mutant monkey precursor in which a single monkey residue (Glu141) was converted to the mouse residue (His141) yielded a cell line that was approximately 100-fold less sensitive to the toxin and the mutant precursor bound the toxin approximately 12-fold less tightly than the wild-type monkey precursor. Taken together, these results indicate that Glu 141 plays a critical role in toxin binding and toxin sensitivity.