Russell-Harde D, Pu H, Betts M, Harkins R N, Perez H D, Croze E
Department of Protein Biochemistry, Berlex Biosciences Inc., Richmond, California 94804-0099, USA.
J Biol Chem. 1995 Nov 3;270(44):26033-6. doi: 10.1074/jbc.270.44.26033.
The type I interferon (IFN) receptor complex is assumed to be composed of multiple protein subunits. Recently, two proteins have been identified as potential receptor components, both of which share a high degree of structural homology with the immunoglobulin superfamily. One of these proteins, referred to as the human interferon alpha receptor (IFNAR), has been shown to be involved in interferon signal transduction, but it does not bind IFN with high affinity. A second putative receptor protein, named FLP40, has been cloned from human Daudi cells. Transfection of FLP40 into murine NIH 3T3 cells does not result in high affinity IFN binding. In this study, we demonstrate that when expressed in murine L929 cells neither IFNAR nor FLP40 by themselves are capable of binding human IFN-alpha 8. Co-expression of IFNAR and FLP40 results in cells capable of binding IFN-alpha 8 and IFN-alpha 2. Scatchard analysis of binding demonstrated the presence of high (KD 350 pM) and low (KD 4.0 nM) affinity binding sites. Binding of radiolabeled IFN-alpha 8 can be competed with either unlabeled IFN-alpha 8 or a recombinant form of human interferon beta, IFN-beta 1b, but not with IFN-gamma. Ligand binding of IFN-alpha 8 can be inhibited by antibodies directed against IFNAR providing further support for a role for this protein in the formation of a ligand binding site. This is the first demonstration indicating that two previously identified IFN receptor proteins, which individually do not bind type I IFN with high affinity, cooperate in the formation of a type I IFN receptor ligand binding complex.
I型干扰素（IFN）受体复合物被认为由多个蛋白质亚基组成。最近，两种蛋白质被鉴定为潜在的受体成分，它们都与免疫球蛋白超家族具有高度的结构同源性。其中一种蛋白质，被称为人干扰素α受体（IFNAR），已被证明参与干扰素信号转导，但它不能高亲和力地结合IFN。第二种假定的受体蛋白，名为FLP40，已从人Daudi细胞中克隆出来。将FLP40转染到鼠NIH 3T3细胞中不会导致高亲和力的IFN结合。在本研究中，我们证明，当在鼠L929细胞中表达时，单独的IFNAR和FLP40都不能结合人IFN-α8。IFNAR和FLP40的共表达导致细胞能够结合IFN-α8和IFN-α2。结合的Scatchard分析表明存在高亲和力（KD 350 pM）和低亲和力（KD 4.0 nM）结合位点。放射性标记的IFN-α8的结合可以被未标记的IFN-α8或重组形式的人干扰素β（IFN-β1b）竞争，但不能被IFN-γ竞争。针对IFNAR的抗体可以抑制IFN-α8的配体结合，这进一步支持了该蛋白在配体结合位点形成中的作用。这是首次证明，两种先前鉴定的IFN受体蛋白，单独不能高亲和力地结合I型IFN，它们在I型IFN受体配体结合复合物的形成中协同作用。
