Cofano F, Moore S K, Tanaka S, Yuhki N, Landolfo S, Appella E
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1990 Mar 5;265(7):4064-71.
The receptor for mouse interferon gamma (IFN-gamma) was purified from detergent-solubilized plasma membranes of EL-4, a thymoma cell line which expresses a high number of receptors on its cell surface. The purification was carried out by immunoaffinity chromatography using an anti-receptor monoclonal antibody. The purified receptor was subjected to NH2-terminal sequence analysis as well as sequencing of endopeptidase-generated peptides. One of the peptides was found to be identical to a portion of the published amino acid sequence of the human IFN-gamma receptor deduced from cDNA. This information was utilized to construct a mixed-sequence oligodeoxynucleotide probe which permitted the isolation of a full-length cDNA clone coding for the mouse IFN-gamma receptor. The mouse IFN-gamma receptor cDNA is comprised of 105 base pairs of the 5'-untranslated region, an open reading frame coding for a 477-amino acid serine-rich protein having calculated Mr 52,276, and a 3'-untranslated region of 539 base pairs. The receptor is first synthesized as a pre-protein from which a 25-amino acid signal peptide is cleaved. The receptor contains a hydrophobic transmembrane portion near the center of the molecule. Northern blot analysis of various cell lines showed that each contained a single 2.0-kilobase mRNA. A direct correlation between the amount of IFN-gamma receptor mRNA and the level of receptor expressed on the cell surface was observed. The mouse and human IFN-gamma receptors are structurally similar, showing 51% over-all homology in amino acid sequence. Mouse IFN-gamma receptor cDNA when inserted in a mammalian shuttle vector and transfected into COS-7 monkey cells was able to direct the expression of specific binding activity for mouse IFN-gamma.
小鼠γ干扰素（IFN-γ）受体是从EL-4胸腺瘤细胞系的去污剂溶解的质膜中纯化得到的，该细胞系在其细胞表面表达大量受体。纯化过程通过使用抗受体单克隆抗体的免疫亲和层析进行。对纯化的受体进行了氨基末端序列分析以及对内肽酶产生的肽段进行测序。发现其中一个肽段与从cDNA推导的人IFN-γ受体已发表氨基酸序列的一部分相同。利用该信息构建了一个混合序列寡脱氧核苷酸探针，从而分离出编码小鼠IFN-γ受体的全长cDNA克隆。小鼠IFN-γ受体cDNA由5'非翻译区的105个碱基对、编码一个477个氨基酸的富含丝氨酸蛋白（计算的Mr为52,276）的开放阅读框以及539个碱基对的3'非翻译区组成。受体最初作为前体蛋白合成，随后切割掉一个25个氨基酸的信号肽。受体在分子中心附近含有一个疏水跨膜部分。对各种细胞系的Northern印迹分析表明，每个细胞系都含有一条单一的2.0千碱基mRNA。观察到IFN-γ受体mRNA的量与细胞表面表达的受体水平之间存在直接相关性。小鼠和人IFN-γ受体在结构上相似，氨基酸序列总体同源性为51%。当将小鼠IFN-γ受体cDNA插入哺乳动物穿梭载体并转染到COS-7猴细胞中时，能够指导对小鼠IFN-γ的特异性结合活性的表达。
