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 identi fied as potential receptor components, both of which share a high degr ee of structural homology with the immunoglobulin superfamily, One of these proteins, referred to as the human interferon alpha receptor (IF NAR), has been shown to be involved in interferon signal transduction, but it does not bind IFN with high affinity, A second putative recept or protein, named FLP40, has been cloned from human Daudi cells, Trans fection of FLP40 into murine NIH 3T3 cells does not result in high aff inity IFN binding, In this study, we demonstrate that when expressed i n 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 an alysis of binding demonstrated the presence of high (K-D 350 pM) and l ow (K-D 4.0 nM) affinity binding sites, Binding of radiolabeled IFN-al pha 8 can be competed with either unlabeled IFN-alpha 8 or a recombina nt form of human interferon beta, IFN-beta 1b, but not with IFN-gamma. Ligand binding of LFN-alpha 8 can be inhibited by antibodies directed against IFNAR providing further support for a role for this protein i n the formation of a ligand binding site, This is the first demonstrat ion indicating that two previously identified IFN receptor proteins, w hich individually do not bind type I IFN with high affinity, cooperate in the formation of a type I IFN receptor ligand binding complex.