Identification of critical residues in bovine IFNAR-1 responsible for interferon binding.

Interferons have antiviral, antigrowth and immunomodulatory effects. The human type I interferons, IFN-alpha, IFN-beta, and IFN-omega, induce somewhat different cellular effects but act through a common receptor complex, IFNAR, composed of subunits IFNAR-1 and IFNAR-2. Human IFNAR-2 binds all type I IFNs but with lower affinity and different specificity than the IFNAR complex. Human IFNAR-1 has low intrinsic binding of human IFNs but strongly affects the affinity and differential ligand specificity of the IFNAR complex. Understanding IFNAR-1 interactions with the interferons is critical to elucidating the differential ligand specificity and activation by type I IFNs. However, studies of ligand interactions with human IFNAR-1 are compromised by its low affinity. The homologous bovine IFNAR-1 serendipitously binds human IFN-alphas with nanomolar affinity. Exploiting its strong binding of human IFN-alpha2, we have identified residues important for ligand binding. Mutagenesis of any of five aromatic residues of bovine IFNAR-1 caused strong decreases in ligand binding, whereas mutagenesis of proximal neutral or charged residues had smaller effects. These residues were mapped onto a homology model of IFNAR-1 to identify the ligand-binding face of IFNAR-1, which is consistent with previous structure/function studies of human IFNAR-1. The topology of IFNAR-1/IFN interactions appears novel when compared with previously studied cytokine receptors.

Localization of a receptor nonapeptide with a possible role in the binding of the type I interferons.

Interferons (IFNs) in common with other cytokines activate Janus tyrosine kinases and latent STAT transcription factors upon binding to their cell surface receptor. Type I IFNs bind to a receptor composed of two transmembrane polypeptides, IFNAR1 and IFNAR2, which belong to the class II cytokine receptor family that also includes the cellular receptors for IFN-gamma, interleukin-10 and coagulation protease factor VII (tissue factor). The extracellular domain of the type I IFN receptor chain IFNAR1, has four fibronectin type-III sub-domains. Human IFNAR1 has intrinsic weak affinity for type I IFNs and plays an essential role in transmembrane signaling, formation of a high affinity complex with IFN and the modulation of ligand specificity. In order to characterise the ligand binding site on IFNAR1 we analysed the epitope recognized by the anti-IFNAR1 mAb, 64G12, which inhibits the binding and biological activities of both IFN-alpha and IFN-beta. The target peptide recognized by the 64G12 mAb was determined by screening a set of 48 overlapping peptides covering the first two subdomains (residues 23-229) of the extracellular region of IFNAR1. The results of this study show that the peptide (FSSLKLNVY), localized within the first sub-domain (residues 89-97) of IFNAR1, which is recognized by the 64G12 mAb, most likely overlaps a site to which both IFN-alpha and IFN-beta bind in the ligand-receptor complex. Thus, since the 64G12 mAb can neutralize the biological activities of all the type I IFNs tested, we suggest that the target peptide recognized by the 64G12 mAb, is a possible anchorage point on IFNAR1, common to binding of both IFN-alpha and IFN-beta.

Characterization of antihuman IFNAR-1 monoclonal antibodies: epitope localization and functional analysis.

The type I interferon receptor (IFNAR) is composed of two subunits, IFNAR-1 and IFNAR-2, encoding transmembrane polypeptides. IFNAR-2 has a dominant role in ligand binding, but IFNAR-1 contributes to binding affinity and to differential ligand recognition. A panel of five monoclonal antibodies (mAb) to human IFNAR-1 (HuIFNAR-1) was produced and characterized. The reactivity of each mAb toward HuIFNAR-1 on native and transfected cells and in Western blot and ELISA formats was determined. In functional assays, one mAb, EA12, blocked IFN-a2 binding to human cells and interfered with Stat activation and antiviral activity. Epitopes for the mAb were localized to subdomains of the HuIFNAR-1 extracellular domain by differential reactivity of the mAb to a series of human/bovine IFNAR-1 chimeras. The antibody EA12 seems to require native HuIFNAR-1 for reactivity and does not map to a single subdomain, perhaps recognizing an epitope containing noncontiguous sequences in at least two subdomains. In contrast, the epitopes of the non-neutralizing mAb FB2, AA3, and GB8 mapped, respectively, to the first, second, and third subdomains of HuIFNAR-1. The mAb DB2 primarily maps to the fourth subdomain, although its reactivity may be affected by other determinants.

Mapping human interferon-alpha (IFN-alpha 2) binding determinants of the type I interferon receptor subunit IFNAR-1 with human/bovine IFNAR-1 chimeras.

Type I interferons bind to a common receptor (IFNAR), composed of two transmembrane polypeptides, IFNAR-1 and IFNAR-2. Although human IFNAR-1 has a weak intrinsic affinity for human Type I interferons (IFNs), bovine IFNAR-1 binds human Type I IFNs with moderate (nM) affinity, and can be conveniently used to investigate the regions of IFNAR-1 involved in ligand binding. We have constructed 14 bovine/human IFNAR-1 chimeras by exchanging homologous subdomains in the extracellular portion of the receptor. These chimeras were expressed at very high levels on COS cells, and their ability to bind HuIFN-alpha2 was measured. No single bovine subdomain substituted into human IFNAR-1 could confer moderate-affinity ligand binding on the resulting chimera. Simultaneous substitution of bovine IFNAR-1 subdomains 2 and 3 for the homologous human subdomains resulted in a dramatic increase in the binding of IFN-alpha2, suggesting that critical determinants for moderate-affinity ligand binding by BoIFNAR-1 reside in these two subdomains. Bovine subdomains 1 and/or 4 each further enhanced IFN-alpha2 binding in the presence of bovine subdomains 2 and 3. Thus, the binding interactions of BoIFNAR-1 with IFNs appears to be more complex than that of other class II cytokine receptors with their ligands.

Bovine type I interferon receptor protein BoIFNAR-1 has high-affinity and broad specificity for human type I interferons.

The type I interferon receptor (IFNAR) is composed of two transmembrane polypeptides, IFNAR-1 and IFNAR-2. Human IFNAR-1 has low intrinsic affinity for IFNs, but enhances the affinity for IFNs of the complex over that of HuIFNAR-2 alone, and modulates the ligand specificity. Bovine cells respond to human alpha interferons. The bovine homologue of HuIFNAR-1, BoIFNAR-1, when expressed in heterologous cells, confers high-affinity binding and broad specificity for human type I IFNs. A soluble fusion protein of the ectodomain of BoIFNAR-1 and an immunoglobulin Fc domain was produced. In contrast to HuIFNAR-1, this protein competes strongly with human cells for IFN binding, and directly binds a wide spectrum of human type I IFNs, including diverse IFN-alphas, IFN-beta and IFN-omega, with moderate to high affinity. This accounts for much of the specificity for human IFNs possessed by bovine cells, with several exceptions. The BoIFNAR-1 ectodomain, in contrast to HuIFNAR-1, may be useful for studies of binary and ternary complexes with IFNs and IFNAR-2, and for purification, assay and biological neutralization protocols.

Contributions of cloned type I interferon receptor subunits to differential ligand binding.

The human type I interferons, including at least 12 IFN-alphas, IFN-beta and IFN-omega, bind to a receptor (IFNAR) composed of at least two transmembrane subunits, IFNAR-1 and IFNAR-2. The contributions of the receptor subunits to ligand binding were investigated by measuring the binding properties of IFNAR-1 or IFNAR-2 alone, or when co-expressed. The affinity of IFNAR-2 for IFN-alpha2 was increased by the co-expression of IFNAR-1, which itself binds ligand very weakly. Most type I IFNs inhibited the binding of IFN-alpha2 to IFNAR-2 alone with IC50 values of 2-20 nM. For cells co-expressing IFNAR-1 and IFNAR-2, the IC50 values decreased 3-20-fold for various ligands, relative to their values on IFNAR-2 alone. Thus, while IFNAR-2 plays the major role in affinity determination and differential recognition of type I IFNs, IFNAR-1 modulates both the ligand affinity and selectivity of the IFNAR-1/IFNAR-2 receptor complex.

Intrinsic ligand binding properties of the human and bovine alpha-interferon receptors.

The Type I interferon receptor (IFN-alpha R) interacts with all IFN-alpha s, IFN-beta and IFN-omega, and seems to be a multisubunit receptor. To investigate the role of a cloned receptor subunit (IFN-alpha R1), we have examined the intrinsic ligand binding properties of the bovine and human IFN-alpha R1 polypeptides expressed in Xenopus laevis oocytes. Albeit with different efficiencies, Xenopus oocytes expressing either the human or bovine IFN-alpha R1 polypeptide exhibit significant binding and formation of crosslinked complexes with human IFN-alpha A and IFN-alpha B. Thus, the IFN-alpha R1 polypeptide most likely plays a direct role in ligand binding.

Expression of a functional human type I interferon receptor in hamster cells: application of functional yeast artificial chromosome (YAC) screening.

The previously cloned human interferon alpha/beta (Hu-IFN-alpha/beta; Type I interferon) receptor cDNA appears to be only one component of a receptor complex since expression of the cDNA in mouse cells confers sensitivity only to Hu-IFN-alpha B2, but a monoclonal antibody against this cloned receptor subunit inhibits biological activities of Hu-IFN-alpha A, Hu-IFN-alpha B2, Hu-IFN-omega, and Hu-IFN-beta. Here we report that a yeast artificial chromosome (YAC) containing a segment of human chromosome 21 introduced into Chinese hamster ovary (CHO) cells confers upon these cells a greatly enhanced response to Hu-IFN-alpha A and Hu-IFN-alpha B2 as well as an increased response to Hu-IFN-omega, Hu-IFN-alpha A/D(Bgl), andd Hu-IFN-beta. These responses were measured by induction of class I MHC antigens and by protection against encephalomyocarditis virus and vesicular stomatitis virus. Furthermore, these cells exhibit specific high affinity binding of Hu-IFN-alpha A and Hu-IFN-alpha B2, Hu-IFN-beta, and Hu-IFN-omega. The results indicate that all the genes necessary to reconstitute a biologically active Type I human IFN receptor complex are located within the human DNA insert of this YAC clone.

Radiation inactivation of human gamma-interferon: cellular activation requires two dimers.

gamma-Interferon (IFN-gamma) is a 17-kDa broad-spectrum cytokine which exerts its effects on a variety of target cells through its interaction with the IFN-gamma receptor. Although physicochemical studies of Escherichia coli-derived IFN-gamma, as well as its crystal structure, demonstrate that it is a homodimer in solution (M(r) 34,000), previous radiation inactivation studies yielded a functional size for IFN-gamma of 63-73 kDa in an antiviral assay. To understand the relationship between the solution form of IFN-gamma and the moiety that actually binds to the cellular receptor and activates cells, we examined irradiated nonradioactive and 32P-labeled IFN-gamma for its migration in SDS/polyacrylamide gels (to determine its physical integrity), its binding to cells, its reactivity in an ELISA, and its antiviral activity. The functional size of IFN-gamma differed in the assays, being 22 +/- 2 kDa for the physical destruction of IFN-gamma, 56 +/- 2 kDa for the cellular binding assay, 45-50 kDa for reactivity in the ELISA, and 72 +/- 6 kDa for antiviral activity. The results from the binding assays constitute direct evidence that IFN-gamma binds to its cellular receptor as a dimer. However, for antiviral activity, the functional mass is equivalent to a tetramer. This is consistent with models involving ligand-induced receptor dimerization, whereby two dimers acting in concert (equivalent to the target size of a tetramer) are required to activate cells in the antiviral assay.

Generation and characterization of anti-idiotypic antibodies recognizing the interferon-alpha receptor: implications for ligand-receptor interactions.

Monoclonal antibodies LI-1 and LI-8 against interferon-alpha A (IFN-alpha A) block IFN-alpha A activity and binding to its receptor, but they recognize distinct epitopes. Surprisingly, anti-idiotypic antibodies to both LI-1 and LI-8 have properties consistent with recognition of the receptor: anti-LI-1 and anti-LI-8 antibodies inhibit the binding of IFN-alpha A to its receptor. However, anti-LI-1 is an antagonist of IFN-alpha A, while anti-LI-8 is an agonist. Thus, at least some part of the epitopes on IFN-alpha A recognized by LI-1 and LI-8 are directly involved in receptor binding. Because these epitopes are spatially distinct, the implication is that the receptor binding site on IFN-alpha A must be extensive, or there are minimally two regions of IFN-alpha A involved in receptor interactions.

Cloning and characterization of a bovine alpha interferon receptor.

A bovine interferon alpha receptor (BoIFN-alpha R1) cDNA, homologous to the human cDNA, was isolated. Transfection of the BoIFN-alpha R1 cDNA into monkey COS cells results in a large increase in high-affinity binding sites for human IFN-alpha A and IFN-alpha B. Covalent crosslinking of radiolabeled HuIFN-alpha A and -alpha B demonstrates that the complex of [32P]HuIFN with the BoIFN-alpha R1 protein (predicted mass, 61,375) expressed in COS cells migrates as a 140-150 kDa band.

Somatic cell mapping of the bovine interferon-alpha receptor.

The bovine interferon-alpha receptor (BoIFN-alpha R) mediates the activity of bovine IFN-alpha s and IFN-beta. In addition, human IFN-alpha s have uniformly high biological activity on bovine cells. A 32P-labeled derivative of human recombinant IFN-alpha A (HuIFN-alpha A-P1) binds well and can form a characteristic 130-kDa complex on bovine cells, but not on hamster cells. We have, therefore, analyzed the binding and covalent crosslinking of [32P]HuIFN-alpha A-P1 to a panel of bovine-hamster somatic cell hybrids. Binding to several bovine-hamster hybrid cell lines was strong (about 30-50% of that seen with bovine MDBK cells) and specific. The binding correlated uniquely with bovine syntenic group U10. In several of the hybrid lines, the ability of human IFN-alpha B to enhance the expression of endogenous MHC class I molecules correlated with the binding results. We thus conclude that the bovine IFN-alpha R structural gene (locus designation IFNAR) localizes to syntenic group U10. This group includes a number of other genes whose homologs map to human Chromosome (Chr) 21.

Detection and analysis of interferon-alpha receptors on plasma membranes and in detergent extracts.

The binding of interferons-alpha (IFN-alpha) to various cells is well characterized, but fewer studies have reported on the interaction of IFN-alpha with receptors on plasma membranes or in detergent-solubilized form. We describe a simple, sensitive, and semiquantitative assay procedure to detect the presence of IFN-alpha receptors on bovine spleen plasma membrane preparations or in detergent-solubilized extracts. The procedure involves spotting the sample on hydrophobic polyvinylidene difluoride (PVDF; Immobilon P) membranes, blocking the filter with milk, and binding radiolabeled IFN-alpha A to the membrane filter, with detection by either autoradiography or scintillation counting. This assay procedure has been applied for the identification of IFN-alpha receptors in crude and affinity-purified fractions. The partially purified IFN-alpha receptors have been further characterized by SDS-polyacrylamide gel electrophoresis (PAGE). The separated IFN-alpha receptor protein on the SDS-PAGE gel has been electrophoretically transferred to Immobilon membrane and visualized by ligand blotting. This provides an estimate of 95-110 kD for the apparent molecular weight and a tool for further studies of the receptor protein.

Sublocalization on chromosome 21 of human interferon-alpha receptor gene and the gene for an interferon-gamma response protein.

The cellular responses to alpha and beta interferons (IFN-alpha and -beta) are mediated through the IFN-alpha/beta (type I) receptor, while the response to IFN-gamma is mediated through the IFN-gamma (type II) receptor. The receptors for IFN-alpha/beta and IFN-gamma are encoded by genes on human chromosomes 21 and 6q, respectively. The presence of chromosome 21q confers both ligand binding and responsiveness to human IFN-alpha/beta, whereas chromosome 6q confers binding of Hu-IFN-gamma, but not cellular responsiveness on somatic cell hybrids. Chromosome 6q (i.e., the Hu-IFN-gamma receptor gene) and chromosome 21q are both necessary for the cellular response of somatic cell hybrids (from fibroblasts) to Hu-IFN-gamma. It is conceivable that the factor mediating activity through the IFN-gamma receptor is, in fact, the IFN-alpha receptor, or that the two genes are distinct but part of an "interferon response" region. Here we more precisely localize on human chromosome 21 the genes for the IFN-alpha receptor and for the factor(s) mediating the action of IFN-gamma through the chromosome 6-encoded receptor. Hamster-human somatic cell hybrids containing various fragments of human chromosome 21 were used. The presence of the human IFN-alpha/beta receptor was determined by binding 32P-labeled human IFN-alpha to cells, covalently cross-linking the [32P]IFN-alpha-receptor complex, and analyzing it by SDS-polyacrylamide gel electrophoresis. The presence of the IFN-gamma receptor-related factor mediating cellular responsiveness was determined by HLA induction in hybrid cells containing the IFN-gamma receptor (chromosome 6q), a transfected copy of the human HLA-B7 gene, and various portions of chromosome 21. In all hybrids examined, the two genes cosegregate. Specifically, both genes are localized to the region of chromosome 21 containing the markers D21S58, D21S65, and GART and appear to be proximal to D21S58. The implications for IFN action are discussed.

Modulation by interferons of HLA antigen, high-molecular-weight melanoma associated antigen, and intercellular adhesion molecule 1 expression by cultured melanoma cells with different metastatic potential.

The effect of leukocyte (IFN-alpha), fibroblast (IFN-beta), and immune (IFN-gamma) interferon and/or mezerein on the expression of HLA antigens and melanoma-associated antigens by the melanoma cell line MeWo and its metastatic variant MeM 50-10 was investigated, since this information may contribute to our understanding of the molecular mechanism(s) underlying the metastatic process and of the role of cell differentiation and growth suppression in the antigenic changes induced by interferon (IFN). The three types of IFN had no effect on the expression of high-molecular-weight melanoma-associated antigen, but enhanced that of HLA Class 1 antigens and of intercellular adhesion molecule 1 on MeWo and MeM 50-10 cells. The enhancing effect of IFN-gamma was more marked than that of IFN-alpha and IFN-beta. Furthermore IFN-gamma enhanced the expression of intercellular adhesion molecule 1 by MeM 50-10 cells more than by MeWo cells. IFN-beta was shown for the first time to induce HLA Class II antigens; the effect of IFN-beta, like that of IFN-gamma, is differential on the two cell lines and on the gene products of the HLA-D region. Like IFN-gamma, IFN-beta induced only HLA-DR antigens on MeM 50-10 cells. The results of Northern blot analysis with HLA-DR beta, -DQ beta, and -DP beta probes suggest that the differential modulation of the gene products of the HLA-D region by IFN-beta and IFN-gamma reflects transcriptional and posttranscriptional events. The differential susceptibility to modulation by IFN-beta and IFN-gamma of HLA Class II antigens on MeWo and MeM 50-10 cells is an intrinsic property of each cell line, since only small differences were detected in the number and/or affinity of receptors on the two cell lines. Furthermore, the lack of marked effects of mezerein on the antigen-modulating activity of the three types of IFN, in spite of an enhancement of their differentiating activity, suggests that the changes in the antigenic profile induced by IFN do not represent a differentiation-related phenomenon.

Construction and phosphorylation of a fusion protein Hu-IFN-alpha A/gamma.

A protein consisting of human (Hu)-IFN-alpha A to which the COOH-terminal 16 amino acids of Hu-IFN-gamma were fused was prepared by constructing an expression vector by oligonucleotide-directed mutagenesis. The hybrid protein Hu-IFN-alpha A/gamma was expressed under the control of phage lambda PL promoter. The protein was purified with the use of a monoclonal antibody against Hu-IFN-alpha or the COOH-terminal amino acid sequence of Hu-IFN-gamma. The purified protein exhibited a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has antiviral activity on human and bovine cells. Unlike Hu-IFN-alpha A, but similar to Hu-IFN-gamma, the hybrid Hu-IFN-alpha A/gamma can be phosphorylated by [gamma 32P]ATP and cAMP-dependent protein kinase. The phosphorylated molecule binds to the IFN-alpha/beta receptor. The introduction of a phosphorylation site into Hu-IFN-alpha A by fusion of the region of Hu-IFN-gamma which contains the phosphorylation site provides a new reagent for studies of receptor binding, pharmacokinetics, and other studies where labeled interferons are useful. Furthermore, the introduction of phosphorylation sites into proteins provides a new principle for the preparation of a wide variety of reagents for many purposes.

Creation of phosphorylation sites in proteins: construction of a phosphorylatable human interferon alpha.

A phosphorylation site was introduced into human interferon alpha A (IFN-alpha A) by site-specific mutation of the coding sequence. Three slightly different phosphorylation sites were created by using the predicted amino acid consensus sequences for phosphorylation by the cAMP-dependent protein kinase. The resultant modified interferons (IFN-alpha A-P) were expressed in Escherichia coli and purified. The purified proteins exhibit antiviral activity on bovine and human cells similar to that of the unmodified IFN-alpha A. The IFN-alpha A-P proteins can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high specific activity (2000-5000 Ci/mmol; 1 Ci = 37 GBq) with retention of biological activity. The 32P-labeled IFN-alpha A-P proteins bind to cells and can be covalently bound to the IFN-alpha/beta receptor with a bifunctional reagent as can human IFN-alpha A. The introduction of phosphorylation sites into proteins provides a procedure to prepare a large variety of radioactive proteins for research and clinical use.