GPI-anchored diphtheria toxin receptor allows membrane translocation of the toxin without detectable ion channel activity.
We have investigated the role of the transmembrane and cytoplasmic domains of the diphtheria toxin (DT) receptor [heparin-binding epidermal growth factor (HB-EGF) precursor] in the intoxication pathway. Two mutants were constructed in which these domains were replaced by either a 37 amino acid sequence signalling membrane attachment via a glycosylphosphatidylinositol (GPI) anchor (DTR-GPI) or by the transmembrane and cytoplasmic domains of the human EGF receptor (DTR-EGFR). Similar amounts of DTA fragment were translocated through the plasma membrane of NIH 3T3 cells transfected with the wild-type receptor (DTR), DTR-GPI and DTR-EGFR, but translocation was about six times less efficient in the case of DTR-GPI and DTR-EGFR when taking into account the number of receptors expressed. Interestingly, DT-induced 22Na+ influx was weak in DTR-EGFR cells and not detectable in DTR-GPI cells. Whole cell patch-clamp analysis showed the DT at low pH induced depolarization and decreased input resistance in DTR cells (and to a lesser extent also in DTR-EGFR cells) but not in DTR-GPI cells. These results suggest that the transmembrane and cytoplasmic part of the receptor might be involved in channel activity and that translocation of the A fragment is independent of toxin-induced cation channel activity.
Lanzrein
M
M
Institute for Cancer Research, Norwegian Radium Hospital, Oslo, Norway.
Sand
O
O
Olsnes
S
S
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D018958:CD55 Antigens / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004167:Diphtheria Toxin / Q000378:metabolism*; D066246:ErbB Receptors / Q000737:chemistry; D017261:Glycosylphosphatidylinositols; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D015640:Ion Channel Gating; D051379:Mice; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship
D018958:CD55 Antigens; D017931:DNA Primers; D004167:Diphtheria Toxin; D017261:Glycosylphosphatidylinositols; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors