The cytoplasmic domain of the diphtheria toxin receptor (HB-EGF precursor) is not required for receptor-mediated endocytosis.
Diphtheria toxin is believed to enter toxin-sensitive mammalian cells by receptor-mediated endocytosis employing the transmembrane cell surface precursor of heparin-binding epidermal growth factor-like growth factor as a receptor. To investigate the contribution of the receptor's cytoplasmic domain to the toxin internalization process, we constructed stable cell lines that express receptor molecules containing cytoplasmic domain mutations. Our results indicate that Tyr192 and surrounding amino acid residues are important for high toxin sensitivity. Cells expressing mutant receptors are less sensitive to toxin and have fewer toxin-specific binding sites but internalize toxin at rates similar to those of cells expressing the intact receptor. This rate of internalization is much slower (1-2%/min) than that of classical endocytic receptors (10-50%/min). Our results are consistent with a model in which the cytoplasmic domain of the toxin receptor lacks a signal for rapid internalization. We suggest that toxin-receptor complexes, nevertheless, are internalized by receptor-mediated endocytosis by entrapment in clathrin-coated pits as part of bulk phase turnover of cell surface proteins. Although the rate is slow, successful intoxication occurs because a single internalized enzymatically-active toxin molecule is sufficient to inhibit protein synthesis in the cell.
Almond
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Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
Eidels
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D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D004167:Diphtheria Toxin / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D014709:Vero Cells
D004167:Diphtheria Toxin; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor