Search

Ligand-induced cross-linking of the type I interferon (IFN) receptor subunits ifnar1 and ifnar2 induces a pleiotrophic cellular response. Several studies have suggested differential signal activation by flexible recruitment of the accessory receptor subunit ifnar1. We have characterized the roles of the four Ig-like sub-domains (SDs) of the extracellular domain of ifnar1 (ifnar1-EC) for ligand recognition and receptor assembling. Various sub-fragments of ifnar1-EC were expressed in insect cells and purified to homogeneity. Solid phase binding assays with the ligands IFN(alpha)2 and IFN(beta) revealed that all three N-terminal SDs were required and sufficient for ligand binding, and that IFN(alpha)2 and IFN(beta) compete for this binding site. Cellular binding assays with different fragments, however, highlighted the key role of the membrane-proximal SD for the formation of an in situ IFN-receptor complex. Even substitution with the corresponding SD from homologous cytokine receptors did not restore high-affinity ligand binding. Receptor assembling analysis on supported lipid bilayers in vitro revealed that the membrane-proximal SD controls appropriate orientation of the receptor on the membrane, which is required for efficient association of ifnar1 into the ternary complex.

Previous studies have reported that mesenchymal stem cells (MSC) may be isolated from the synovial membrane by the same protocol as that used for synovial fibroblast cultivation, suggesting that MSC correspond to a subset of the adherent cell population, as MSC from the stromal compartment of the bone marrow (BM). The aims of the present study were, first, to better characterize the MSC derived from the synovial membrane and, second, to compare systematically, in parallel, the MSC-containing cell populations isolated from BM and those derived from the synovium, using quantitative assays. Fluorescent-activated cell sorting analysis revealed that both populations were negative for CD14, CD34 and CD45 expression and that both displayed equal levels of CD44, CD73, CD90 and CD105, a phenotype currently known to be characteristic of BM-MSC. Comparable with BM-MSC, such MSC-like cells isolated from the synovial membrane were shown for the first time to suppress the T-cell response in a mixed lymphocyte reaction, and to express the enzyme indoleamine 2,3-dioxygenase activity to the same extent as BM-MSC, which is a possible mediator of this suppressive activity. Using quantitative RT-PCR these data show that MSC-like cells from the synovium and BM may be induced to chondrogenic differentiation and, to a lesser extent, to osteogenic differentiation, but the osteogenic capacities of the synovium-derived MSC were significantly reduced based on the expression of the markers tested (collagen type II and aggrecan or alkaline phosphatase and osteocalcin, respectively). Transcription profiles, determined with the Atlas Human Cytokine/Receptor Array, revealed discrimination between the MSC-like cells from the synovial membrane and the BM-MSC by 46 of 268 genes. In particular, activin A was shown to be one major upregulated factor, highly secreted by BM-MSC. Whether this reflects a different cellular phenotype, a different amount of MSC in the synovium-derived population compared with BM-MSC adherent cell populations or the impact of a different microenvironment remains to be determined. In conclusion, although the BM-derived and synovium-derived MSC shared similar phenotypic and functional properties, both their differentiation capacities and transcriptional profiles permit one to discriminate the cell populations according to their tissue origin.

Type I Interferons (IFN) are key cytokines in the regulation of the innate immune response. All IFNs bind to a shared cell surface receptor comprised of two subunits, IFNAR1 and IFNAR2. Together with IFN the receptors assemble to hetero-trimeric “ternary” complexes which initiate phosphorylation cascades through the intracellularly associated Janus-Kinases Jak1 and Tyk2. Detailed structure–function analysis of IFNs has established that the dynamics and affinities of IFN interactions with the receptor subunits play a critical role for regulating signalling specificities. Here we have explored the IFN-induced assembly of the ternary signalling complex at physiological expression level by dual colour single molecule imaging. To this end, we employed highly specific and efficient orthogonal posttranslational labelling approaches with photostable organic fluorophores combined with TIRF-microscopy. By single molecule co-localization and co-tracking analysis we have mapped lateral distribution and diffusion of the two receptor subunits with very high spatial and temporal resolution in living cells. Thus, we were able to monitor IFN-induced assembly, co-diffusion and dissociation of individual ternary signalling complexes in the plasma membrane. Systematic modulation of the binding affinities towards the receptor subunits revealed that binding affinity of IFNα2 towards IFNAR1 is optimized for efficient ternary complex formation at physiological receptor surface concentrations. Interestingly, the negative feedback regulator ubiquitin-specific protease 18 (USP18) reduces receptor dimerization by IFNα2, but not IFNβ, which binds to IFNAR1 with higher affinity. Strikingly, this USP18-dependent loss of 2D affinity could be mimicked by truncating the receptor’s intracellular domains. These observations suggest that ternary complexes are stabilized by intracellular interactions of Jak1 and Tyk2 and that these interactions are interfered by USP18, thus selectively abrogating signalling by IFNs with low affinity towards IFNAR1.

The four mammalian Jak tyrosine kinases are non-covalently associated with cell surface receptors binding helical bundled cytokines. In the type I interferon receptor, Tyk2 associates with the IFNAR1 receptor subunit and positively influences ligand binding to the receptor complex. Here, we report that Tyk2 is essential for stable cell surface expression of IFNAR1. In the absence of Tyk2, mature IFNAR1 is weakly expressed on the cell surface. Rather, it is localized into a perinuclear endosomal compartment which overlaps with that of recycling transferrin receptors and with early endosomal antigen-1 (EEA1) positive vesicles. Conversely, co-expressed Tyk2 greatly enhances surface IFNAR1 expression. Importantly, we demonstrate that Tyk2 slows down IFNAR1 degradation and that this is due, at least in part, to inhibition of IFNAR1 endocytosis. In addition, Tyk2 induces plasma membrane relocalization of the R2 subunit of the interleukin-10 receptor. These results reveal a novel function of a Jak protein on internalization of a correctly processed cytokine receptor. This function is distinct from the previously reported effect of other Jak proteins on receptor exit from the endoplasmic reticulum.

Nature 517, 89–93 (2015); doi:10.1038/nature13801In this Letter, author J.-L.C. was inadvertently missing one of their affiliations; they should also have been associated with affiliation number 2 (St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, New York 10065, USA). This has been corrected in the online versions of the paper.

Type I IFN regulates different aspects of the immune response, inducing a cell-mediated immunity. We have recently shown that the infection of dendritic cells (DC) with Mycobacterium tuberculosis (Mtb) induces IFN-α. In this work we have monitored a rapid induction of IFN-β followed by the delayed production of the IFN-α1 and/or -α13 subtypes. The Mtb infection rapidly activates the NF-κB complex and stimulates the phosphorylation of IFN regulatory factor (IRF)-3, events known to induce IFN-β expression in viral infection. In turn, the autocrine production of IFN-β induces the IFN-stimulated genes that contain binding sites for activated STATs in their promoters. Among the IFN-stimulated genes induced in DC through STAT activation are IRF-1 and IRF-7. The expression of IRF-1 appears to be dependent on the sequential activation of NF-κB and STAT-1. Once expressed, IRF-1 may further stimulate the transcription of IFN-β. Induction of IRF-7 is also regulated at the transcriptional level through the binding of phosphorylated STAT-1 and STAT-2, forming the IFN-stimulated gene factor-3 complex. In turn, the IRF-1 and IRF-7 expression appears to be required for the delayed induction of the IFN-α1/13 genes. Although correlative, our results strongly support the existence of a cascade of molecular events in Mtb-infected DC. Upon infection, constitutively expressed NF-κB and IRF-3 are activated and likely contribute to the rapid IFN-β expression. In turn, IFN-β-induced IRF-1 and IRF-7 may cooperate toward induction of IFN-α1/13 if infection persists and these factors are activated.

International audience; Tyrosine kinases of the Janus kinase family initiate cellular responses through their association with receptors for alpha-helical cytokines. In addition to a tyrosine kinase domain, these enzymes possess a kinase-like (KL) domain, whose function remains elusive. To investigate the role of the KL domain of Tyk2 in interferon-alpha/beta signaling, we transfected a library of Tyk2 cDNAs containing random point mutations in KL into Tyk2-negative cells and selected for loss-of-function Tyk2 mutants. Four such mutants, V584D, G596V, H669P, and R856G, were identified through this screen. Like the wild-type Tyk2, the mutant proteins were able to sustain the level of IFNAR1 receptor protein. However, all four mutants were incapable of restoring high-affinity interferon-alpha binding in Tyk2-negative cells and were also catalytically impaired, even when transiently overexpressed, Interferon-alpha induced phosphorylation, and gene expression could be detected in V584D- or G596V-expressing cells, but not in H669P- or R856G-expressing cells. Furthermore, H669P and R856G proteins were constitutively highly phosphorylated. All together, our findings demonstrate that an intact KL domain is essential for the intrinsic catalytic activity of Tyk2 and for the establishment of a high-affinity interferon-alpha receptor complex.

The cytokine receptor (CR) family is structurally defined by the presence in the extracellular domain of its members of a typical 200-amino-acid domain (D200). This domain is built of two subdomains of 100 amino acids (SD100A and SD100B) that are both structured like the constant domain of the immunoglobulins (Bazan 1990; Thoreau et al. 1991). Conserved cysteines allow the definition of class I and class II CR and clearly distinguish the CR family from the fibronectin type III family and the immunoglobulin superfamily. The CR family is also genetically defined by the intron/exon structures encoding the D200: All members of the family have introns conserved both in position and in phase (Lutfalla et al. 1992; Nakagawa et al. 1994). The genes encoding the members of the CR family are scattered all over the human genome. When clusters are observed, they encode proteins that are structurally the most similar, and where the orientation of transcription of the genes in the clusters is known, they are transcribed in the same orientation (Gorman et al. 1992; Kremer et al. 1993; Lutfalla et al. 1995). This suggests that the family has evolved through successive rounds of duplications, that the oldest duplications have been scattered all over the genome, but that the most recent ones are still conserved in tandem. This evolution is probably a vertebrate story because no CR has ever been reported in any invertebrate species. The CRs are part of the most rapidly evolving category of proteins, that is, the “host defense ligands and receptors” (Murphy 1993). Class I members have been described in fishes (Sandra et al. 1995), birds (Burnside et al. 1991), and mammals, but class II only in mammals. As mainly shown using knockout mice, class II cytokine receptors (CRIIs) are active in various host defence mechanisms: antiviral (α/β or type I interferons) (Muller et al. 1994), antiparasitic (γ or type II interferon) (Huang et al. 1993), tolerance to bowel mucosal antigens [interleukin-10 (IL-10)] (Spencer et al. 1998), and blood coagulation [tissue factor (TF)] (Morrissey et al. 1987). The two components of the receptors for α/β IFN (IFNAR1 and IFNAR2), for γ interferon (IFNGR1 and IFNGR2), and for IL-10 (IL10R1 and IL10R2) together with TF constitute the seven known CRII family members. With the exception of TF, which has virtually no intracellular domain, the genes coding the CRII have a similar intron/exon structure encoding the intracellular domain: They all have a single similarly placed phase 0 intron interrupting the coding region for the intracellular domain (Lutfalla et al. 1992, 1993, 1995; Raval et al. 1995; Rhee et al. 1996). They therefore have a close evolutionary relationship that is also reflected by the fact that four of them are in tandem on human chromosome 21 (Fig ​(Fig1A):1A): IFNAR2, IL10R2, IFNAR1, and IFNGR2. These four genes, together with the GART gene (Schild et al. 1990) that encodes three steps of purine synthesis, form a synteny group that is conserved in human and mouse (Cheng et al. 1993). The close proximity of some genes in this GART–CRII gene cluster (e.g., 500 bp between IFNAR2 and IL10R2) suggests that the large regions between the previously characterized genes could harbor other genes (Fig. ​(Fig.1A)1A) (Lutfalla et al. 1995). Figure 1 Structure of the genomic loci downstream of the GART gene in human, chicken, and Fugu: Genes are indicated with their names, size, and orientation of transcription (arrows). Scale is indicated in each panel. (A) Human locus on chromosome 21. Arrowheads ... Among the three biological systems in which receptors from the GART–CRII gene cluster are involved, the α/β IFN (type I IFN) is of special interest. In contrast to IL-10 and γ IFN (type II IFN) that are encoded by single genes, all species have several type I IFNs. Phylogenetic analysis of these type I IFNs based on nonsynonymous substitution has shown that β IFN diverged from α ∼250 Mya, that is, after the bird/mammal divergence (Hughes 1995). This is confirmed by the fact that the multiple bird type I IFNs can be neither classified as α nor as β (Sekellick et al. 1994; Sick et al. 1996). This major divergence between α and β IFNs early in mammalian evolution is reflected by the fact that they do not interact the same way with their shared receptor components (Lewerenz et al. 1998) and can induce distinct biological responses (Erlandsson et al. 1998). These observations therefore raise the question of the structure of the type I IFN receptor that the mammals inherited from their ancestors and how it managed to cope with such a diversification of its ligands. Outside the mammalian family, the IFN system has been described and molecularly characterized only in birds with most of the efforts concentrated on chicken where the interferon system was first described by Isaacs and Lindenman (1957). The sequences of many different bird IFNs are now available (Sekellick et al. 1994; Digby and Lowenthal 1995), but the structure of their receptors still remains unknown. Chicken was therefore the first nonmammalian species of choice in which to look for the structure of the GART–CRII homologous locus. The pufferfish Fugu rubripes owing to its compact genome is the best suited fish species in which to study the structure of a gene cluster. We decided to use comparative genomic analysis to study how the type I IFN receptor genes developed during the evolution of the GART–CRII gene cluster in vertebrates. For this purpose, we first sequenced the gaps between the already known genes in the human GART–CRII gene cluster to have a complete description of its gene content. We then compared its large-scale structure with that of the chicken and of the pufferfish Fugu rubripes.

International audience; Type I interferon (IFN) subtypes alpha and beta share a common multicomponent, cell surface receptor and elicit a similar range of biological responses, including antiviral, antiproliferative, and immunomodulatory activities. However, alpha and beta IFNs exhibit key differences in several biological properties. For example, IFN-beta, but not IFN-alpha, induces the association of tyrosine-phosphorylated receptor components ifnar1 and ifnar2, and has activity in cells lacking the IFN receptor-associated, Janus kinase tyk2. To define the structural basis for these functional differences we produced human IFN-beta with point mutations and compared them to wild-type IFN-beta in assays that distinguish alpha and beta IFN subtypes. IFN-beta mutants with charged residues (N86K, N86E, or Y92D) introduced at two positions in the C helix lost the ability to induce the association of tyrosine-phosphorylated receptor chains and had reduced activity on tyk2-deficient cells. The combination of negatively charged residues N86E and Y92D (homologous with IFN-alpha 8) increased the cross-species activity of the mutant IFN-beta s on bovine cells to a level comparable to that of human IFN-alpha s. In contrast, point mutations in the AB loop and D helix had no significant effect on these subtype-specific activities. A subset of these latter mutations did, however, reduce activity in a manner analogous to IFN-alpha mutations, The effects of these mutations on IFN-beta activity are discussed in the context of a family of related ligands acting through a common receptor and signaling pathway.

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-α (IFN-α) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-α. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-α receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficientin vitroIFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-α. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.

Systemic toxicity currently prevents full clinical application of type I interferons (IFNs) for the treatment of cancers and viral infections. Although the use of IFNs fused to antibodies resulted in a 10-fold increased activity towards targeted cells, off-target side effects experienced by the patients are expected to remain a major obstacle. We present a novel strategy to engineer IFN α with high in vitro and in vivo activity towards cells expressing a specific surface marker while having an up to 1000-fold reduced activity towards non-relevant cells. The concept lies in the use of immunocytokines based on IFN mutants with strongly reduced binding affinity for the IFN receptor. Such a targeted IFN recovers almost maximally its antiviral activity uniquely on the targeted cells and acts to selected cell populations in mice with targeting efficiencies up to 3 orders of magnitude. This novel concept holds promise to revitalize the clinical potential of IFN α and other cytokines.

A full-length cDNA corresponding to a gene mapping to the D21S58 locus was cloned. The encoded protein, called CRF2-4, was shown to be a typical class II member of the cytokine receptor family. The gene encoding CRF2-4 spans more than 30 kb. Its intron/exon structure was determined and shown to be conserved with all other members of the cytokine receptor family. The physical distance between the CRF2-4 gene and its IFNAR neighbor has been narrowed to less than 35 kb.

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

Using the cDNA coding for the human interferon alpha/beta receptor (IFNAR), the IFNAR gene has been physically mapped relative to the other loci of the chromosome 21q22.1 region. 32,906 base pairs covering the IFNAR gene have been cloned and sequenced. Primer extension and solution hybridization-ribonuclease protection have been used to determine that the transcription of the gene is initiated in a broad region of 20 base pairs. Some aspects of the polymorphism of the gene, including noncoding sequences, have been analyzed; some are allelic differences in the coding sequence that induce amino acid variations in the resulting protein. The exon structure of the IFNAR gene and of that of the available genes for the receptors of the cytokine/growth hormone/prolactin/interferon receptor family have been compared with the predictions for the secondary structure of those receptors. From this analysis, we postulate a common origin and propose an hypothesis for the divergence from the immunoglobulin superfamily.

Two polymorphic loci within the interferon-alpha receptor (IFNAR) gene on human chromosome 21 have been identified and mapped by linkage analysis in 40 CEPH families. These markers are (1) a multiallelic RFLP with an observed heterozygosity of 0.72 and (2) a variable (AT3)n short sequence repeat at the poly(A) tail of an Alu sequence (AluVpA) with an observed heterozygosity of 0.83. This locus is close to D21S58 (theta = 0.02, zeta = 36.76) and D21S17 (theta = 0.02, Zeta = 21.76) with chromosomal band 21q22.1. Multipoint linkage analysis suggests the most likely locus order to be 21cen-D21S58-IFNAR-D21S17-21qter. Given its high heterozygosity, the IFNAR gene can be used as an index marker on human chromosome 21.

The cellular receptor for the human alpha and beta interferons (IFN) was expressed, by gene transfer, in a murine hepatoma-derived cell line, BTG 9A. Injected subcutaneously into the syngeneic mouse (C57BL/6), the parental and the transfected cells grew and formed tumors which later regressed. More than half the mice bearing tumors derived from cells expressing the receptor, developed IgG antibodies capable of blocking the activity, on human cells, of human recombinant IFN-alpha B, -alpha A, -alpha D and of natural human IFN-beta, but not of recombinant IFN-gamma. Cross-reactivity of human IFN-alpha on murine and bovine cells was unaffected by these antibodies. The binding of human IFN-alpha to solubilized receptors from human lymphoid cell lines was also blocked and complexes of radiolabeled recombinant IFN-alpha A or IFN-alpha B, chemically cross-linked to their human receptor could be immunoprecipitated by the antisera. IFN alpha beta receptor protein, purified by electrophoresis in sodium dodecylsulfate, was not recognized. We conclude that the antibodies are directed against the forms of the IFN alpha beta receptor actually expressed on the membrane.

Previous studies have reported that mesenchymal stem cells (MSC) may be isolated from the synovial membrane by the same protocol as that used for synovial fibroblast cultivation, suggesting that MSC correspond to a subset of the adherent cell population, as MSC from the stromal compartment of the bone marrow (BM). The aims of the present study were, first, to better characterize the MSC derived from the synovial membrane and, second, to compare systematically, in parallel, the MSC-containing cell populations isolated from BM and those derived from the synovium, using quantitative assays. Fluorescent-activated cell sorting analysis revealed that both populations were negative for CD14, CD34 and CD45 expression and that both displayed equal levels of CD44, CD73, CD90 and CD105, a phenotype currently known to be characteristic of BM-MSC. Comparable with BM-MSC, such MSC-like cells isolated from the synovial membrane were shown for the first time to suppress the T-cell response in a mixed lymphocyte reaction, and to express the enzyme indoleamine 2,3-dioxygenase activity to the same extent as BM-MSC, which is a possible mediator of this suppressive activity. Using quantitative RT-PCR these data show that MSC-like cells from the synovium and BM may be induced to chondrogenic differentiation and, to a lesser extent, to osteogenic differentiation, but the osteogenic capacities of the synovium-derived MSC were significantly reduced based on the expression of the markers tested (collagen type II and aggrecan or alkaline phosphatase and osteocalcin, respectively). Transcription profiles, determined with the Atlas Human Cytokine/Receptor Array, revealed discrimination between the MSC-like cells from the synovial membrane and the BM-MSC by 46 of 268 genes. In particular, activin A was shown to be one major upregulated factor, highly secreted by BM-MSC. Whether this reflects a different cellular phenotype, a different amount of MSC in the synovium-derived population compared with BM-MSC adherent cell populations or the impact of a different microenvironment remains to be determined. In conclusion, although the BM-derived and synovium-derived MSC shared similar phenotypic and functional properties, both their differentiation capacities and transcriptional profiles permit one to discriminate the cell populations according to their tissue origin.

Using membrane preparations of the interferon receptor, prepared from cells of the Burkitt line, Daudi, we have examined the binding of three human recombinant alpha-interferons. 1. We discovered a binding titration of the interferons IFN-alpha A and IFN-alpha D in the pH range 6-9. Receptor binding, negligible at pH 6, rises to a maximum close to pH 9. We have shown that binding of IFN-alpha A at basic pH is to the same receptors as at neutrality and that IFN-receptor complexes extracted with digitonin are more stable at basic pH than they are at neutrality. 2. The recombinant interferon, IFN-alpha B, shows little change of binding in the pH range 6-9. At its basic optimum the binding of IFN-alpha A approaches that of IFN-alpha B, while at neutral pH the binding of IFN-alpha A is 3-4 times less. This difference at neutral pH is seen on intact cells as well as on membrane preparations. The specific activity of IFN-alpha B is close to that of IFN-alpha A, both of which are 10-20 times more active than IFN-alpha D; and the binding titration is, therefore, independent of the initial binding affinities. 3. Using hybrid IFNs constructed from the DNA sequences of alpha D and alpha B, we have isolated the sequence responsible for the binding titration to the segment comprising amino acids 61-92. Examination of these sequences reveals that Lys-84 is present in all the IFN-alpha except IFN-alpha B where it is replaced by Glu; and Tyr-90, present in most of the common IFN-alpha including alpha A and alpha D, is replaced by Asp in IFN-alpha B. Lys and Tyr would normally titrate in the pH range 6-9. We conclude that the binding titration is due to an electrostatic interaction and we propose that the interaction is between IFN-receptor complexes. The role of the interaction in the binding losses that accompany the antiproliferative effects of IFN is discussed.

The Burkitt-derived line, Daudi, whose proliferation is inhibited by human alpha-interferon (IFN-alpha), was treated with 125I-labelled recombinant human IFN-alpha A. After separation from unbound ligand, cell-bound IFN was extracted with the detergent digitonin yielding soluble and insoluble complexes of IFN and receptor, together with a certain amount of uncomplexed IFN. 1. Soluble complexes were stable enough to be separated from uncomplexed IFN by permeation chromatography. Treatment of soluble complexes with the bifunctional reagent, disuccinimidyl suberate, yielded a radioactive product separating with an Mr of 130,000 on electrophoresis in sodium dodecyl sulphate. Similar complexes could be recovered with sodium dodecyl sulphate from the digitonin-insoluble residue, treated with the bi-functional reagent. 2. The total (soluble and insoluble) of complexed IFN obtained after digitonin extraction was a constant fraction (0.62) of the total cell-bound radioactivity, being independent of the concentration of IFN added to the cells (less than pM to greater than nM), and of the time of incubation (1 min to 20 h). However, between 30 min and 3 h of incubation, the insoluble complex increased, at the expense of the soluble complex, and there appeared a cellular pool of degraded ligand. From 3 h to 20 h the distribution of ligand-derived radioactivity remained constant while the total amount decreased to less than 10% of its value at 30 min. This decrease in binding was matched by the appearance of an equivalent quantity of radiolabelled fragments in the culture medium. 3. The inhibition of cellular division due to IFN was shown to be coincident with the disappearance of cellular binding and with the cell-mediated degradation of receptor-complexed IFN. We propose that IFN removes its own receptor and, in doing so, blocks a linked function necessary for the stimulated growth of Daudi cells.

Two human recombinant lymphoblastoid interferon-alpha subtypes, LyIFN-B (alpha 8) and LyIFN-D (alpha 1), and 10 hybrids generated therefrom were produced in Escherichia coli and purified. The antiviral and antiproliferative activities and the induction of (2'-5')oligoadenylate synthetase were compared to their receptor binding affinities. The IFN subtypes and their hybrids had similar specific antiviral activities on bovine cells. On human cells both the specific antiviral and antiproliferative activities of LyIFN-B were about 30-fold higher than those of LyIFN-D. This difference in activity could be attributed partly to the N-terminal amino acids 1 to 60 and partly to amino acids 61 to 92. A third domain affecting the biological activities was found within the carboxy-proximal segment from amino acids 93 to 150. The differences in these activities were found to correlate with their ability to bind the receptor, suggesting that the differences in activity might be due to altered binding of the IFNs to the cellular receptors. In contrast, the induction of (2'-5')oligoadenylate synthetase did not follow the same activity profile. On mouse cells, the efficiency of the hybrids was affected by at least four sites on the IFN protein. A hybrid with the N-terminal segment 1 to 60 from IFN-B and amino acids 61 to 166 from IFN-D had a specific antiviral activity on mouse cells as high as on human cells corresponding to a 500- and 5000-fold increase in specific activity compared to IFN-D and IFN-B, respectively. We suggest that on mouse cells the IFN activity may be more dependent on conformational differences than on human cells, which in turn might reflect a less precise fit to the mouse receptor than to the human receptor.

Evidence is presented, based on sequence comparison according to Hydrophobic Cluster Analysis, of a structural and evolutionary relationship between the human alpha/beta-interferon receptor and the human and mouse gamma-interferon receptor. These results predict that the human alpha/beta-interferon receptor extracellular part is organised in two homologous subdomains connected by a proline linker. They also predict that both subdomains present some homologies to the external domain of mouse and human gamma interferon receptor.

Publisher Summary This chapter discusses the radioiodination of human alpha interferons by the chloramines T method. The chloramine T method has long been used for incorporating iodine into the tyrosine residues of proteins. The reactants generated are strong oxidants, and for sensitive proteins gentler methods may be needed. The chloramine T reaction involves a slow hydrolysis to generate hypochlorite followed by fast reactions with iodide to form oxidized species that substitute rapidly at positions ortho to the tyrosine hydroxyl, the first substitution facilitating the second. The iodine atom has dimensions similar to those of the phenolate ion, so substitution would likely lead to local distortions in protein structure. The method was originally devised to keep hypochlorite and molecular iodine concentrations as low as possible during the reaction, thus reducing damage and the escape of volatile iodine. In a properly screened and ventilated hood, the reaction poses no great hazard, but particular care is needed to avoid contamination because, in the method described, iodide is allowed to oxidize before it is allowed to react.

The human interferon-α receptor gene (IFN AR) has been assigned to the long arm of human chromosome 21 (report of the committee on the genetic constitution of chromosomes 20 and 21; Ref 1). The present report confirms the assignment and refines the mapping to the 21q22.1 band, using a cDNA probe for the human IFN AR gene and in situ hybridization to metaphase chromosomes.