Wietzerbin J, Gaudelet C, Aguet M, Falcoff E
J Immunol. 1986 Apr 1;136(7):2451-5.
Recombinant E. coli-derived murine IFN-gamma (Mu-rIFN-gamma; 5 X 10(7) U/mg) was radiolabeled with 125I by the chloramine-T method without loss of its antiviral activity. The 125I-Mu-rIFN-gamma showed specific binding to L1210 cells. Scatchard analysis indicates about 4000 binding sites per cell and an apparent Kd of 5 X 10(-10)M. Binding of 125I-Mu-rIFN-gamma to cells was inhibited by both natural (glycosylated) and rIFN-gamma, but not by IFN-alpha/beta. Receptor-bound 125I-Mu-rIFN-gamma was rapidly internalized when incubation temperature was raised from 4 degrees C to 37 degrees C. On internalization, almost no IFN-gamma degradation was observed during 16 hr incubation. 125I-Mu-rIFN-gamma binding capacity decreased in cells preincubated with low doses of unlabeled Mu-rIFN-gamma, but not with IFN-alpha/beta. This receptor down-regulation was dose-dependent: 90% reduction of 125I-Mu-rIFN-gamma binding was observed after preincubation with 100 U/ml. After removal of IFN-gamma from the culture medium, the binding capacity increased with time. However, reappearance of receptor was completely blocked by cycloheximide or tunicamycin, suggesting that re-expression of receptors is not due to recycling but to the synthesis of new receptors, and that the receptor is probably a glycoprotein. Cross-linking of 125I-Mu-rIFN-gamma to surface L1210 cell proteins by using bifunctional agents yielded a predominant complex of m.w. 110,000 +/- 5000. Thus, assuming a bimolecular complex, the m.w. of the receptor or receptor subunit would be close to 95,000 +/- 5000. The formation of such a complex appeared highly specific on the basis of the following criteria: it could be inhibited by the addition of Mu-rIFN-gamma but not by Mu-rIFN-alpha/beta, it was not obtained in cells pretreated with IFN-gamma to induce down-regulation of IFN-gamma receptors, and it was also identified in the IFN-alpha/beta-resistant L1210R cell line, known to be sensitive to IFN-gamma and which we have recently shown to express IFN-gamma receptors.
通过氯胺 - T法用125I对重组大肠杆菌衍生的小鼠干扰素 - γ（Mu - rIFN - γ；5×10⁷U/mg）进行放射性标记，其抗病毒活性未丧失。125I - Mu - rIFN - γ显示出与L1210细胞的特异性结合。Scatchard分析表明每个细胞约有4000个结合位点，表观解离常数为5×10⁻¹⁰M。天然（糖基化）和重组IFN - γ均可抑制125I - Mu - rIFN - γ与细胞的结合，但IFN - α/β则不能。当孵育温度从4℃升高到37℃时，受体结合的125I - Mu - rIFN - γ迅速内化。内化后，在16小时的孵育过程中几乎未观察到IFN - γ降解。用低剂量未标记的Mu - rIFN - γ预孵育的细胞中，125I - Mu - rIFN - γ的结合能力降低，但IFN - α/β预孵育的细胞则不然。这种受体下调是剂量依赖性的：用100 U/ml预孵育后，观察到125I - Mu - rIFN - γ结合减少90%。从培养基中去除IFN - γ后，结合能力随时间增加。然而，受体的重新出现被环己酰亚胺或衣霉素完全阻断，这表明受体的重新表达不是由于再循环而是由于新受体的合成，并且该受体可能是一种糖蛋白。通过使用双功能试剂将125I - Mu - rIFN - γ与表面L1210细胞蛋白交联产生了一个主要的分子量为110,000±5000的复合物。因此，假设形成双分子复合物，受体或受体亚基的分子量将接近95,000±5000。基于以下标准，这种复合物的形成显得高度特异性：加入Mu - rIFN - γ可抑制其形成，但Mu - rIFN - α/β则不能；在用IFN - γ预处理以诱导IFN - γ受体下调的细胞中未获得该复合物；并且在已知对IFN - γ敏感且我们最近已证明表达IFN - γ受体的IFN - α/β抗性L1210R细胞系中也鉴定到了该复合物。
