Imamura K, Sherman M L, Spriggs D, Kufe D
Laboratory of Clinical Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1988 Jul 25;263(21):10247-53.
Tumor necrosis factor (TNF) is a monokine that induces pleiotropic events in both transformed and normal cells. These effects are initiated by the binding of TNF to high affinity cell surface receptors. The post-receptor events and signaling mechanisms induced by TNF, however, have remained unknown. The present studies demonstrate the presence of a single class of high affinity receptors on membranes prepared from HL-60 promyelocytic leukemic cells. The interaction of TNF with these membrane receptors was associated with a 3.8-fold increase in specific binding of the GTP analogue, GTP gamma S. Scatchard analysis of GTP gamma S binding data demonstrated that TNF stimulates GTP binding by increasing the affinity of available sites. The TNF-induced stimulation of GTP binding was also associated with an increase in GTPase activity. Moreover, the increase in GTPase activity induced by TNF was sensitive to pertussis toxin. The results also demonstrate that TNF similarly increased GTP binding and pertussis toxin-sensitive GTPase activity in membranes from mouse L929 fibroblasts, thus indicating that these effects are not limited to hematopoietic cells. Analysis of HL-60 membranes after treatment with pertussis toxin in the presence of [32P]NAD revealed three substrates with relative molecular masses of approximately Mr 41,000, 40,000, and 30,000. In contrast, L929 cell membranes had only two detectable pertussis toxin substrates of approximately Mr 41,000 and 40,000. Although the Mr 41,000 pertussis toxin substrate represents the guanine nucleotide-binding inhibitory protein Gi, the identities of the Mr 40,000 and Mr 30,000 substrates remain unclear. In any event, inhibition of the TNF-induced increase in GTPase activity and ADP-ribosylation of Gi by pertussis toxin suggested that TNF might act by increasing GTPase activity of the Gi protein. However, the results further indicate that TNF has no detectable effect on basal or prostaglandin E2-stimulated cAMP levels in HL-60 cells. Taken together, these findings indicate that a pertussis toxin-sensitive GTP-binding protein other than Gi, and possibly the Mr 40,000 substrate, is involved in the action of TNF. Finally, the demonstration that pertussis toxin inhibited TNF-induced cytotoxicity in L929 cells supports the presence of a GTP-binding protein which couples TNF-induced signaling to a biologic effect.
肿瘤坏死因子（TNF）是一种单核因子，可在转化细胞和正常细胞中引发多种效应。这些效应是由TNF与高亲和力细胞表面受体结合引发的。然而，TNF诱导的受体后事件和信号传导机制仍不清楚。目前的研究表明，在从HL-60早幼粒细胞白血病细胞制备的膜上存在一类单一的高亲和力受体。TNF与这些膜受体的相互作用与GTP类似物GTPγS的特异性结合增加3.8倍有关。对GTPγS结合数据的Scatchard分析表明，TNF通过增加可用位点的亲和力来刺激GTP结合。TNF诱导的GTP结合刺激也与GTP酶活性增加有关。此外，TNF诱导的GTP酶活性增加对百日咳毒素敏感。结果还表明，TNF同样增加了小鼠L929成纤维细胞膜中的GTP结合和百日咳毒素敏感的GTP酶活性，因此表明这些效应不限于造血细胞。在用[32P]NAD存在下的百日咳毒素处理HL-60膜后进行分析，发现了三种相对分子质量约为41,000、40,000和30,000的底物。相比之下，L929细胞膜只有两种可检测到的相对分子质量约为41,000和40,000的百日咳毒素底物。尽管相对分子质量为41,000的百日咳毒素底物代表鸟嘌呤核苷酸结合抑制蛋白Gi，但相对分子质量为40,000和30,000的底物的身份仍不清楚。无论如何，百日咳毒素对TNF诱导的GTP酶活性增加和Gi的ADP核糖基化的抑制表明，TNF可能通过增加Gi蛋白的GTP酶活性来发挥作用。然而，结果进一步表明，TNF对HL-60细胞中的基础或前列腺素E2刺激的cAMP水平没有可检测到的影响。综上所述，这些发现表明，除了Gi之外，一种对百日咳毒素敏感的GTP结合蛋白，可能还有相对分子质量为40,000的底物，参与了TNF的作用。最后，百日咳毒素抑制L929细胞中TNF诱导的细胞毒性的证明支持了存在一种将TNF诱导的信号传导与生物学效应偶联的GTP结合蛋白。
