O'Neill L A, Bird T A, Gearing A J, Saklatvala J
Cytokine Biochemistry Group, Strangeways Research Laboratory, Worts Causeway, Cambridge, United Kingdom.
J Biol Chem. 1990 Feb 25;265(6):3146-52.
The post-receptor events which follow the binding of interleukin 1 (IL1) to cells are unclear. The present studies provide evidence for the activation of a guanine nucleotide binding protein (G protein) by IL1 in the membranes of an IL1 receptor-rich strain (NOB-1) of the EL4 murine thymoma line. IL1 alpha and beta increased the binding of the GTP analogue [35S]guanosine 5'-[gamma-thiol]trisphosphate (GTP gamma S) to membranes prepared from these cells. By 1 min after addition of IL1 there was a 2-fold enhancement in binding which was dose dependent in the range 0.1-100 ng/ml. A qualitatively similar result was obtained with IL1 beta although it was 10 times less potent. Specific neutralizing antisera to IL1 alpha and IL1 beta abolished the response. Experiments in which the concentration of [35S]GTP gamma S was varied revealed that IL1 increased the affinity of the binding sites for [35S]GTP gamma S and not their number. IL1 alpha was shown to stimulate GTPase activity in the membranes, the time and concentration dependence of this was similar to that observed for increased [35S]GTP gamma S binding. Half-maximal enhancement of [35S]GTP gamma S binding by IL1 alpha, measured after 4 min, occurred at 5% IL1 receptor occupancy. Maximal stimulation was achieved when 30% of receptors were occupied. Experiments with pertussis and cholera toxins revealed that pretreating membranes with pertussis toxin (100 ng/ml) inhibited by 50% the IL1-induced [35S]GTP gamma S binding and [gamma-32P]GTP hydrolysis. Cholera toxin (100 ng/ml) was without effect. However, both pertussis and cholera toxins at concentrations of 100 ng/ml inhibited IL1-induced IL2 secretion in EL4 NOB-1 cells. These results show that the IL1 receptor of a responsive thymoma line activates, and may be coupled to, a G protein(s). This is a possible mechanism of IL1 signal transduction.
白细胞介素1（IL1）与细胞结合后发生的受体后事件尚不清楚。目前的研究为EL4小鼠胸腺瘤系中富含IL1受体的菌株（NOB-1）膜中IL1激活鸟嘌呤核苷酸结合蛋白（G蛋白）提供了证据。IL1α和β增加了GTP类似物[35S]鸟苷5'-[γ-硫醇]三磷酸（GTPγS）与这些细胞制备的膜的结合。加入IL1后1分钟，结合增加了2倍，在0.1-100 ng/ml范围内呈剂量依赖性。IL1β也得到了定性相似的结果，但其效力低10倍。针对IL1α和IL1β的特异性中和抗血清消除了该反应。改变[35S]GTPγS浓度的实验表明，IL1增加了[35S]GTPγS结合位点的亲和力，而不是其数量。已证明IL1α刺激膜中的GTP酶活性，其时间和浓度依赖性与观察到的[35S]GTPγS结合增加相似。4分钟后测量，IL1α使[35S]GTPγS结合增加到最大值的一半时，IL1受体占有率为5%。当30%的受体被占据时达到最大刺激。用百日咳毒素和霍乱毒素进行的实验表明，用百日咳毒素（100 ng/ml）预处理膜可抑制IL1诱导的[35S]GTPγS结合和[γ-32P]GTP水解50%。霍乱毒素（100 ng/ml）无作用。然而，浓度为100 ng/ml的百日咳毒素和霍乱毒素均抑制EL4 NOB-1细胞中IL1诱导的IL2分泌。这些结果表明，反应性胸腺瘤系的IL1受体激活并可能与一种或多种G蛋白偶联。这是IL1信号转导的一种可能机制。
