Reconstitution of a high affinity binding site for type I interferons.
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.
Russell-Harde
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Department of Protein Biochemistry, Berlex Biosciences Inc., Richmond, California 94804-0099, USA.
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Betts
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Harkins
R N
RN
Perez
H D
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Croze
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D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D016898:Interferon-alpha / Q000378:metabolism; D016899:Interferon-beta / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D007700:Kinetics; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000378:metabolism* / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D016898:Interferon-alpha; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D053633:Receptor, Interferon alpha-beta; D016899:Interferon-beta; D007371:Interferon-gamma