Diphtheria toxin receptor sites on membranes of cultured cells and erythrocytes demonstrated by fluorescence and electron microscopy.
Diphtheria toxin (DT) receptor sites were identified on cell membranes by indirect immunofluorescence and immunoferritin labelling. The results obtained were correlated (1) with the binding of 125I-labelled DT (to the very same cell populations used in the ultrastructural studies), and (2) with toxin sensitivity as shown by inhibition of 14C-amino acid incorporation into protein. Binding and immunocytochemical studies were carried out at 0 degrees C to preclude internalization of DT. Receptor sites for DT were observed on functionally responsive, cultured human diploid (BUD-8) fibroblasts as well as on a small fraction of erythrocytes (RBC) of DT-susceptible species, i.e. human and sheep. On all BUD-8 cell sections DT receptor sites were located in annular patches and not on microvilli as had been observed in the KB epithelial cell line. Immunoferritin label was revealed on only 10% +/- 2.5 (SD) of human RBC in discreet loci along the cell membrane. Specificity of binding of DT to human fibroblast and RBC receptor sites was further demonstrated by competition between 125I-labeled and non-labelled DT. There was no specific binding to DT-treated cultured mouse L929 fibroblasts or to RBC of mouse of rat, species resistant to the toxin. Thus, direct ultrastructural immunocytochemical evidence is provided for the presence of specific cellular DT receptors on cultured cells and erythrocytes of species sensitive to DT, but not on those of species resistant to DT.
D000818:Animals; D002462:Cell Membrane / Q000032:analysis* / Q000648:ultrastructure; D002478:Cells, Cultured; D004167:Diphtheria Toxin / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000032:analysis; D005347:Fibroblasts / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells / Q000032:analysis; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007739:L Cells / Q000032:analysis; D051379:Mice; D008854:Microscopy, Electron; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000032:analysis* / Q000378:metabolism
D004167:Diphtheria Toxin; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; C587885:Hbegf protein, rat; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic