Ligand-Induced Association of the Type I Interferon Receptor Components

Cohen, Batya; Novick, Daniela; Barak, Sara; Rubinstein, Menachem

doi:10.1128/mcb.15.8.4208

Cited by 161 publications

(120 citation statements)

References 30 publications

(28 reference statements)

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“…Because IFN binds to both ifnar1 and ifnar2 on the cell surface, the binding affinity toward ifnar2 is enhanced by cooperative binding of ifnar1. Thus, typically a 10 -100-fold higher affinity constant is observed for IFN binding to the cell surface receptor than for ifnar2 alone (35). The constant offset in Equation 1 most likely accounts for this constant energetic contribution of ifnar1 to form the ternary complex, and the value p AV is in good agreement with the relative contribution of ifnar1.…”

Section: Discussionsupporting

confidence: 68%

New Structural and Functional Aspects of the Type I Interferon-Receptor Interaction Revealed by Comprehensive Mutational Analysis of the Binding Interface

Piehler¹,

Roisman

Schreiber

2000

Journal of Biological Chemistry

140

163

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Type I interferons bind to two cell surface receptors, ifnar1 and ifnar2, as the first step in the activation of several signal transduction pathways that elicit an antiviral state and an anti-proliferative response. Here, we quantitatively mapped the complete binding region of ifnar2 on interferon (IFN)␣2 by 35 individual mutations to alanine and isosteric residues. Of the six "hot-spot" residues identified (Leu-30, Arg-33, Arg-144, Ala-145, Met-148, and Arg-149), four are located on the E-helix, which is located at the center of the binding site flanked by residues on the A-helix and the AB-loop. The contribution of residues of the D-helix, which have been previously implicated in binding, proved to be marginal for the interaction with the extracellular domain of ifnar2. Interestingly, the ifnar2 binding site overlaps the largest continuous hydrophobic patch on IFN␣2. Thus, hydrophobic interactions seem to play a significant role stabilizing this interaction, with the charged residues contributing toward the rapid association of the complex. Relating the anti-viral and anti-proliferative activity of the various interferon mutants with their affinity toward ifnar2 results in linear function over the whole range of affinities investigated, suggesting that ifnar2 binding is the rate-determining step in cellular activation. Dose-time analysis of the anti-viral response revealed that shortening the incubation time of low-level activation cannot be compensated by higher IFN doses. Considering the strict dependence of the cellular response on affinity, these results suggest that for maintaining transcription of IFN-responsive genes over a longer time period, low but continuous signaling through the IFN receptor is essential. Type I interferons (IFN)1 are a family of homologous cytokines, which potently elicit an anti-viral and anti-proliferative state in cells. All human type I IFNs (IFN␣, ␤, , and ) bind to a cell surface receptor consisting of two transmembrane proteins, ifnar1 (1) and ifnar2 (2), which associate upon binding. Binding of IFN to its receptor mediates activation of numerous genes (3) through different signal transduction pathways (reviewed in Ref. 4). Intriguingly, different expression profiles are induced by different IFNs, apparently through the same receptor, with particular differences between IFN␣ and IFN␤ (3, 5). However, the molecular mechanisms underlying the functional differences between IFNs are still unknown. Type I IFNs belong to the class of helical cytokines (6) and are built by five helices. The structure of several type I IFNs have been resolved, such as murine IFN␤ (7), human IFN␣2 (8, 9), human IFN␤ (10), and ovine IFN (11). Mutational studies have revealed functionally important residues on IFNs (reviewed in Ref. 12), which can be ascribed to distinct functional epitopes that interact with ifnar1 and ifnar2 (13, 14). The binding site for ifnar2 was mainly mapped on the AB-loop and D-helix and the binding site for ifnar1 on the C-helix. However, comparison with homologous cytokines...

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Section: Discussionsupporting

confidence: 68%

New Structural and Functional Aspects of the Type I Interferon-Receptor Interaction Revealed by Comprehensive Mutational Analysis of the Binding Interface

Piehler¹,

Roisman

Schreiber

2000

Journal of Biological Chemistry

140

163

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“…However, after longer incubation times the fraction of apoptotic cells induced by YNS closely matches that of IFN␤. 4 YNS Is a Stronger ISG Inducer Than IFN␤-We have previously shown that the HEQ mutant induces a similar gene expression profile as IFN␤ (18). Here, we wanted to determine whether the optimized YNS mutant could further increase the induction of ISG expression.…”

Section: The Higher Antiproliferative Potency Of Yns Is Reflected By mentioning

confidence: 99%

“…All type I interferons induce their biological effects through a common receptor, composed of two distinct transmembrane proteins, namely IFNAR1 and IFNAR2 (3)(4)(5). Upon formation of the ternary complex, the interferon signal is transduced through receptor-associated Janus family kinases (JAK), which activate the signal transducers and activators of transcription (STAT) proteins.…”

mentioning

confidence: 99%

An Interferon α2 Mutant Optimized by Phage Display for IFNAR1 Binding Confers Specifically Enhanced Antitumor Activities

Kalie¹,

Jaitin²,

Abramovich

et al. 2007

Journal of Biological Chemistry

122

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All ␣-interferons (IFN␣) bind the IFNAR1 receptor subunit with low affinity. Increasing the binding affinity was shown to specifically increase the antiproliferative potency of IFN␣2. Here, we constructed a phage display library by randomizing three positions on IFN␣2 previously shown to confer weak binding to IFNAR1. The tightest binding variant selected, comprised of mutations H57Y, E58N, and Q61S (YNS), was shown to bind IFNAR1 60-fold tighter compared with wild-type IFN␣2, and 3-fold tighter compared with IFN␤. Binding of YNS to IFNAR2 was comparable with wild-type IFN␣2. The YNS mutant conferred a 150-fold higher antiproliferative potency in WISH cells compared with wild-type IFN␣2, whereas its antiviral activity was increased by only 3.5-fold. The high antiproliferative activity was related to an induction of apoptosis, as demonstrated by annexin V binding assays, and to specific gene induction, particularly TRAIL. To determine the potency of the YNS mutant in a xenograft cancer model, we injected it twice a week to nude mice carrying transplanted MDA231 human breast cancer cells. After 5 weeks, no tumors remained in mice treated with YNS, whereas most mice treated with wild-type IFN␣2 showed visible tumors. Histological analysis of these tumors showed a significant antiangiogenic effect of YNS, compared with wild-type IFN␣2. This work demonstrates the application of detailed biophysical understanding in the process of protein engineering, yielding an interferon variant with highly increased biological potency. Type I interferons (IFNs)3 are multifunctional cytokines that orchestrate the antiviral innate immunity in vertebrates, acting in virtually every nucleated cell. They belong to the helical cytokine superfamily (1), and consist of at least 17 different members in humans: 13 different IFN␣ subtypes, IFN␤, IFN, and IFN, all of which share significant amino acid homology (2).All type I interferons induce their biological effects through a common receptor, composed of two distinct transmembrane proteins, namely IFNAR1 and IFNAR2 (3-5). Upon formation of the ternary complex, the interferon signal is transduced through receptor-associated Janus family kinases (JAK), which activate the signal transducers and activators of transcription (STAT) proteins. These, in turn, form homo-and heterodimers that translocate to the nucleus, where they directly regulate the transcription of specific interferon responsive genes (6).In addition to their important roles at the first line of defense against viral infections (7) and in modulation of the adaptive immune system (8), type I interferons are capable of inducing a significant antiproliferative activity (9). Consequently, interferons are widely used in the clinic for the treatment of multiple sclerosis and chronic hepatitis (10, 11), as well as for a number of malignancies (12). However, the efficiency of interferon treatment in the majority of cancers is modest, limited by side effects and varying resistance of malignant cells to this treatment (13,14).Despite their...

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“…Peptide Synthesis-The chicken ovalbumin peptide 323-339 (20), phosphorylated and nonphosphorylated peptides of the human IFN-␣/␤ R1 (14) and R2 (21), and the human Stat4-Y694 were synthesized on an Applied Biosystems' peptide synthesizer, model 430 (Foster City, CA . hIFN); FACS, fluorescence-activated cell sorter; EMSA, electrophoretic mobility shift assay; SH2, Src homology 2; STAT, signal transducers and activators of transcription.…”

Section: Methodsmentioning

confidence: 99%

Recruitment of Stat4 to the Human Interferon-α/β Receptor Requires Activated Stat2

Farrar¹,

Smith

Murphy

et al. 2000

Journal of Biological Chemistry

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Ligand-Induced Association of the Type I Interferon Receptor Components

Cited by 161 publications

References 30 publications

New Structural and Functional Aspects of the Type I Interferon-Receptor Interaction Revealed by Comprehensive Mutational Analysis of the Binding Interface

New Structural and Functional Aspects of the Type I Interferon-Receptor Interaction Revealed by Comprehensive Mutational Analysis of the Binding Interface

An Interferon α2 Mutant Optimized by Phage Display for IFNAR1 Binding Confers Specifically Enhanced Antitumor Activities

Recruitment of Stat4 to the Human Interferon-α/β Receptor Requires Activated Stat2

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