Almond B D, Eidels L
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1994 Oct 28;269(43):26635-41.
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.
白喉毒素被认为是通过受体介导的内吞作用进入对毒素敏感的哺乳动物细胞的，该过程利用肝素结合表皮生长因子样生长因子的跨膜细胞表面前体作为受体。为了研究受体胞质结构域在毒素内化过程中的作用，我们构建了稳定表达含有胞质结构域突变的受体分子的细胞系。我们的结果表明，酪氨酸192及其周围的氨基酸残基对于高毒素敏感性很重要。表达突变受体的细胞对毒素的敏感性较低，毒素特异性结合位点较少，但毒素内化速率与表达完整受体的细胞相似。这种内化速率比经典的内吞受体（10-50%/分钟）慢得多（1-2%/分钟）。我们的结果与一种模型一致，即毒素受体的胞质结构域缺乏快速内化的信号。我们认为，毒素-受体复合物仍然是通过受体介导的内吞作用，作为细胞表面蛋白质整体周转的一部分，被困在网格蛋白包被的小窝中而内化的。虽然速率很慢，但成功的中毒仍会发生，因为单个内化的酶活性毒素分子就足以抑制细胞中的蛋白质合成。
