DIPHTHERIA-TOXIN ENDOCYTOSIS AND MEMBRANE TRANSLOCATION ARE DEPENDENT ON THE INTACT MEMBRANE-ANCHORED RECEPTOR (HB-EGF PRECURSOR) - STUDIES ON THE CELL-ASSOCIATED RECEPTOR CLEAVED BY A METALLOPROTEASE IN PHORBOL-ESTER-TREATED CELLS

Preincubation of Vero cells with 1 mu M phorbol 12-myristate 13-acetate (PMA) decreased the specific binding of diphtheria toxin by about 50%, whereas the toxic effect, endocytic uptake and membrane translocation were completely blocked. Toxin bound to PMA-treated cells was released upon incubation with heparinase. The effect of PMA was abrogated in the presence of EDTA or N-{DL-[2-(hydroxyaminocarbonyl)methyl]-4- pentanoyl}-L-3-(2'-naphthyl)-alanyl-L-alanine 2-aminoethylamide (TAPI), a specific inhibitor of matrix metalloproteases. The results indicate that PMA induces proteolytic cleavage of the diphtheria-toxin receptor [heparin-binding EGF-like growth factor (HB-EGF)-precursor] outside the membrane anchor, and that about 50% of the growth-factor ecto-domain remains associated with the cells, due to binding to surface proteoglycans containing heparan sulphates. Although the cleaved cell-associated HB-EGF binds diphtheria toxin, it does not serve as a functional receptor, since neither toxin internalization nor translocation occurs. Thus the intact HB-EGF precursor is of crucial importance for its function as the diphtheria-toxin receptor.