title: GPI-anchored diphtheria toxin receptor allows membrane translocation of the toxin without detectable ion channel activity.
abstract: 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.
authors.affiliation: Institute for Cancer Research, Norwegian Radium Hospital, Oslo, Norway.
authors.forename: M
authors.initials: M
authors.lastname: Lanzrein
authors.forename: O
authors.initials: O
authors.lastname: Sand
authors.forename: S
authors.initials: S
authors.lastname: Olsnes
mesh_terms: 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
chemical_list: 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