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 microM 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-methyl-pentanoyl)-L-3-(2' - naphthyl)-alanyl-L-alanine 2-aminoethyl-amide (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.
Lanzrein
M
M
Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
Garred
O
O
Olsnes
S
S
Sandvig
K
K
D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D004167:Diphtheria Toxin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D019763:Heparin Lyase; D066257:Heparin-binding EGF-like Growth Factor; D006868:Hydrolysis; D036341:Intercellular Signaling Peptides and Proteins; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D011133:Polysaccharide-Lyases / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014709:Vero Cells
D004167:Diphtheria Toxin; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011956:Receptors, Cell Surface; D008666:Metalloendopeptidases; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase; D013755:Tetradecanoylphorbol Acetate