Cloning and Expression of a Long Form of the β Subunit of the Interferon αβ Receptor That Is Required for Signaling

Abstract

The interferon ␣␤ receptor (IFN␣R) or type I IFN-R is formed by a 110-kDa ␣ subunit or IFNAR and by a ␤ subunit, which has short and long forms (molecular masses of 55 and 95-100 kDa, respectively). In this report, we demonstrate that the IFN␣/␤R cDNA recently cloned corresponds to the 55-kDa or short form of the ␤ subunit, while the 95-100-kDa species reported here corresponds to a longer form of the IFN␣/␤R cDNA that is probably produced by alternative splicing of the same gene. Stable transfection of the ␣ subunit with either form of the ␤ subunit results in the expression of low and high affinity receptors, while expression of either form of the ␤ subunit alone only produces low affinity receptors. More important, only expression of the ␣ and long form of the human ␤ subunits in mouse L-929 cells reconstitutes the activation of the Jak kinases and the Stat factors, as well as the antiviral response to human type I IFNs.Characterization of the interferon ␣␤ receptor or type I interferon receptor (IFN␣R or IFN-R) 1 with monoclonal antibodies (mAb) has revealed that this receptor is composed of at least two chains: the ␣ and ␤ subunits (recognized by the IFNaR3 and IFNaR␤1 monoclonal antibodies, respectively) (1, 2). Using mAbs and affinity cross-linking methods, we described two forms of the type I IFN-R: normal and variant (2, 3). The "normal" receptor is expressed in most cells and is composed of ␣ and ␤ subunits with molecular masses of 110 and 100 kDa, respectively. The "variant" receptor is expressed in monocytic cell lines and normal bone marrow cells, and in contrast to the "normal" receptor, its ␤ subunit has a molecular mass of 55 kDa (2, 3). Two cDNAs encoding IFNAR (4) and IFN␣␤R (5) subunits of the type I IFN-R have been cloned. We have recently demonstrated that the ␣ subunit is encoded by the IFNAR cDNA (6), and its cytoplasmic domain directly interacts with Tyk-2 tyrosine kinase.The IFN␣␤R cDNA product (5) has a similar M r as the variant form of the ␤ subunit (2) and is proposed to form a disulfide-bonded dimer that it is only cleaved by high concentrations of reducing agents (5 Cloning of the Long Form of the ␤ Subunit-A cDNA library was made with poly(A)ϩ RNA obtained from the human myeloma U-266 cell line using a commercial cDNA kit, followed by subcloning into the pcDNA II vector (Invitrogen). Screening was performed using a IFN␣/␤R cDNA (5) probe. Positive colonies were obtained after three rounds of screening, and the inserts were mapped using restriction endonucleases and polymerase chain reaction. Both strands of the longest cDNA clone (4A1) were sequenced using an automated sequencing machine (373A DNA sequencer, Applied Science Laboratories) by the method of DyeDeoxy Terminator, Cycle Sequencing Kit (Applied). Partial sequences of shorter cDNA clones were also obtained using the same method. Expression of the Different Type I IFN-R Subunits in Mouse L-929Cells-Mouse L-929 cells were transfected using Lipofectamine Reagent (Life Technologies, Inc.) with the ␤ S , ␤ L , and ␣ subunit...

The human papilloma virus (HPV)-18 E6 oncoprotein physically associates with Tyk2 and impairs Jak-STAT activation by interferon-α

“…The IFN-a receptor is present at low numbers on the surface of all cell types and consists of two transmembrane proteins named IFNAR1 and IF-NAR2-2 (Novick et al, 1994;Uze et al, 1990). The IFNAR2 gene generates several alternatively spliced forms, but only the product harboring a long intracytoplasmic domain (IFNAR2-2) is part of a functional IFN-a receptor (Domanski et al, 1995;Lutfalla et al, 1995) (Figure 9). A membrane-proximal 33 amino acid sequence of the cytoplasmic domain of IFNAR1 physically associated with Tyk2 (Colamonici et al, 1994a,b) (Figure 9A).…”

Homodimerization and intermolecular tyrosine phosphorylation of the Tyk‐2 tyrosine kinase

“…et al [23] have demonstrated that mutant U1D cells bind IFNc~ with low affinity, and that transfection of Tyk-2 restores high affinity binding. The low affinity binding observed in U 1D cells [23] resembles the low affinity receptors observed in mouse transfectants that only express the human fl subunit of the type I IFN-R [7]. These findings led us to hypothesize that an interaction, either direct or through adapter proteins, between the Tyk-2 and Jak-1 kinases brings together the ~ and fl subunits to form the high affinity receptor.…”

“…The role of intermolecular phosphorylation of the homodimer is not clear, but it is possible that it serves as an amplification system which activates neighboring receptors. In a second stage, Tyk-2 and Jak-1 tyrosine phosphorylate the c~ and fl subunit of the receptor, respectively [7,8], and the Statl and Stat2 transcription factors. Our preliminary studies indicate that the Statl and Star2 factors are docked to the flL subunit in a ligand-independent fashion forming a constitutive complex with the receptor and Jak kinases (Domanski et al, manuscript in preparation).…”

“…The ~ subunit corresponds to the cDNA termed IFNAR cloned by Uz6 et al [4,5]. The IFNc~flR cDNA recently cloned by Novick et al [6] corresponds to a short form of the fl subunit (termed fls), while a long form (termed ilL) has been recently cloned in our laboratory and is reported elsewhere [7]. The cytoplasmic domain of the ~ subunit directly associates with Tyk-2 [5,8].…”

Regulated proteolysis of the IFNaR2 subunit of the interferon-alpha receptor

“…The JAKs bind constitutively to receptor subunits, are activated when ligand binding dimerizes the receptor subunits and sequentially phosphorylate multiple components of the signaling cascade. In the case of the type I IFNs, including IFN-a and -b, ligand binding to the IFNaR1 and IFNaR2 receptor subunits juxtaposes and activates Tyk2 and Jak1, the associated JAK kinases (Colamonici et al, 1994;Domanski et al, 1995;Yan et al, 1996b). The JAKs become phosphorylated, and in turn phosphorylate a critical tyrosine on IFNaR1, which functions as a docking site for the Stat2 SH2 domain (Yan et al, 1996a;Li et al, 1997).…”