Journal of Molecular Biology
Volume 366, Issue 2, 16 February 2007, Pages 525-539
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Differential Receptor Subunit Affinities of Type I Interferons Govern Differential Signal Activation

https://doi.org/10.1016/j.jmb.2006.11.053Get rights and content

Abstract

Type I interferons (IFNs) elicit antiviral, antiproliferative and immunmodulatory responses by binding to a shared cell surface receptor comprising the transmembrane proteins ifnar1 and ifnar2. Activation of differential response patterns by IFNs has been observed, suggesting that members of the family play different roles in innate immunity. The molecular basis for differential signaling has not been identified yet. Here, we have investigated the recognition of various IFNs including several human IFNα species, human IFNω and human IFNβ as well as ovine IFNτ2 by the receptor subunits in detail. Binding to the extracellular domains of ifnar1 (ifnar1-EC) and ifnar2 (ifnar2-EC) was monitored in real time by reflectance interference and total internal reflection fluorescence spectroscopy. For all IFNs investigated, competitive 1:1 interaction not only with ifnar2-EC but also with ifnar1-EC was shown. Furthermore, ternary complex formation was studied with ifnar1-EC and ifnar2-EC tethered onto solid-supported membranes. These analyses confirmed that the signaling complexes recruited by IFNs have very similar architectures. However, differences in rate and affinity constants over several orders of magnitude were observed for both the interactions with ifnar1-EC and ifnar2-EC. These data were correlated with the potencies of ISGF3 activation, antiviral and anti-proliferative activity on 2fTGH cells. The ISGF3 formation and antiviral activity correlated very well with the binding affinity towards ifnar2. In contrast, the affinity towards ifnar1 played a key role for antiproliferative activity. A striking correlation was observed for relative binding affinities towards ifnar1 and ifnar2 with the differential antiproliferative potency. This correlation was confirmed by systematically engineering IFNα2 mutants with very high differential antiproliferative potency.

Introduction

Type I interferon (IFN) signaling plays a key role in the innate immune response against viral and bacterial infection as well as malignancies.1 Binding of IFNs to their shared cell surface receptor ifnar1 and ifnar2 subunits activates numerous signal transduction cascades resulting in a pleiotrophic cellular response.2 The cellular response upon treatment with IFNs is typically discriminated into antiviral, antiproliferative and immunmodulatory activities. In comparison to other cytokine receptors the high redundancy of ligands (∼ 15 IFNα subtypes, one IFNβ, one IFNκ, one IFNω in humans)3 binding to the same receptor is unusual, suggesting differential functional properties. Indeed, numerous instances of apparently differential cellular response upon treatment with different IFNs have been reported.4., 5., 6., 7., 8., 9. IFNs are already successfully used for the treatment of rather different diseases,10 and understanding the molecular basis of such differential signaling could tremendously contribute towards systematical engineering of IFNs for medical application. However, the assessment of differential signal activation is extremely difficult for several reasons: different cell types have been used in different studies and for different activity assays, and differential responses appear to be dependent on the cellular context. Furthermore different strategies have been used for defining differential responses. In many cases, antiviral protection and the inhibition of cell proliferation were compared for different IFNs on the level of dose-response curves. While similar, relatively low IFN concentrations are required for antiviral protection, more different, and in some cases also much higher IFN concentrations are required for eliciting antiproliferative response.11 Only a few studies are available where the cellular response was quantified on the mRNA level.12., 13., 14. In these studies differential gene activation was observed at low, but not at high IFN doses, confirming the key role of IFN concentration for modulating the cellular response. All these data taken together suggest that non-linear dose-response and affinity-response behavior, which has also been observed for other cytokine receptors,15 may play a role for differential signal activation.

The interaction of IFNα2 and IFNβ with the high-affinity subunit ifnar2 has been investigated in vitro using the extracellular domain (ifnar2-EC), and extended structure-function studies have been carried out.16., 17., 18., 19., 20., 21. While only minor differences were observed in the structure of the complexes of ifnar2-EC with IFNα2 and IFNβ,22 a ten to 20-fold higher affinity of IFNβ for ifnar2-EC was observed. While the interaction of IFNs with the extracellular domain of ifnar1 (ifnar1-EC) appears rather complex,23., 24. competitive binding and largely overlapping binding epitopes were observed for IFNα2 and IFNβ.25 These binding studies suggested that IFNα2 and IFNβ recruit a 1:1:1 complex with ifnar2 and ifnar1, which probably have rather similar architectures.25 However, a 100-fold higher affinity towards ifnar1-EC was obtained for IFNβ compared to IFNα2.26 Analysis of ligand binding to ifnar1-EC and ifnar2-EC on solid-supported lipid bilayers revealed dynamic equilibria between binary and ternary complexes,26., 27. which are determined by the rate constants of the interaction with the receptor subunits in plane of the membrane. These results suggested that the ternary complex stability and the efficiency of recruitment of ifnar1 into the ternary complex may play a key role for differential signaling and responsiveness. Indeed, the interaction with ifnar1 was shown to be important for absolute and relative antiproliferative activity.20., 28., 29.

Here, we have scrutinized a set of different IFNs with respect to receptor subunit binding and signal activation, namely human IFNα1, IFNα2, IFNα8, IFNα21, IFNβ, and IFNω, as well as ovine IFNτ2 (trophoblastin). Trophoblastins are only found in ruminants, but also activate the human type I interferon receptor,30 and have been reported to exert particularly low antiproliferative acivities.31., 32. Kinetic rate constants and equilibrium dissociation constants were determined by solid phase binding assays with the receptor subunits ifnar1-EC and ifnar2-EC in vitro. Furthermore, assembly of the ternary complex was studied on solid-supported membranes. Correlation with cellular signal activation and responses confirmed the key role of absolute affinity towards ifnar1 for antiproliferative activity. More importantly, however, the relative affinity to the receptor subunits was shown to play a key role for the differential activity of type I IFNs. Based on these insights, we successfully engineered type I IFNs with highly differential activity of IFNα2 mutants.

Section snippets

IFNs have very different affinities towards the receptor subunits

The interaction of the different IFNs with ifnar2-EC and ifnar1-EC was studied by real-time solid phase detection in order to identify differences in affinity and rate constants. The receptor subunits were site-specifically immobilized through their C-terminal decahistidine-tags onto silica surface of the transducer using multivalent chelator heads.33 Non-specific binding of IFNs was negligible at these surfaces after blocking excess immobilization sites with MBP-H10. Binding of IFNs was

Discussion

Induction of differential response patterns by different type I IFNs has been frequently described, but the molecular and biophysical basis of how differential signaling through the same cell surface receptor is achieved has remained enigmatic. Many speculations such as different structures of the ligand-receptor complexes, different orientations of the complexes, different complex stoichiometries and complex compositions, or even additional components have been suggested to be responsible for

Protein biochemistry

IFNβ (formulated Rebif 22 μg and 44 μg) was obtained from Serono GmbH, Unterschleißheim/Germany. IFNα2 and ifnar2-EC with a C-terminal decahistidine-tag were expressed in Escherichia coli, refolded from inclusion bodies and purified by anion exchange and size-exclusion chromatography as described.36 Ifnar1-EC with a C-terminal decahistidine-tag was expressed in Sf9 insect cells and purified from the supernatant by immobilized metal ion affinity and size-exclusion chromatography as described.26

Acknowledgements

IFNβ (Rebif) was generously provided by Dr Garth Virgin, Serono GmbH, Unterschleißheim. The hospitality and the support from the laboratory of Robert Tampé are gratefully acknowledged, in particular excellent technical assistance by Eckhard Linker with cell culture. This project was supported by grants from the Deutsche Forschungsgemeinschaft (Emmy-Noether Program PI 405/1 and the SFB 628) to J.P., and by the Human Frontier Science Program (RGP60/2002) to J.P. and G.S.

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