It is well documented that affinity differences in receptor recognition are at the origin of the specific bioactivities of different IFN subtypes (reviewed in reference 29
). It is unknown, however, how these differences impact the intensity and diversification of signaling cascades, gene expression programs, and biological outputs. To address the mechanistic basis of the IFN-α2/β differential responses, we have measured a number of key intracellular events induced by two IFN subtypes in cells of common origin which differ only in receptor abundance. To measure receptor activation, we quantitated Jak/Stat phosphorylation as the earliest detectable signaling event as well as gene induction, cell cycle lengthening, and apoptosis in a dose-dependent fashion. The main observations of this comparative study are hereafter summarized. In U5-low/low cells, with a twofold-higher level of IFNAR2 than parental 2fTGH cells, the two IFN subtypes are virtually equipotent in Jak/Stat phosphorylation, gene induction, and cell cycle lengthening. However, as seen in WISH cells, IFN-β exhibits a higher antiproliferative potency than IFN-α2, demonstrating that IFN-β is able to bring about a unique change toward the antigrowth effect. In cells with abundant receptors (U5-low/hi and U5-hi/hi), IFN-α2 equals or even becomes more potent than IFN-β in all readouts. These data confirm the inverse correlation between the IFN-α2/β differential and overall IFN sensitivity suggested from comparing cells of different lineages (Fig. ).
How can this be explained mechanistically? Although antiproliferative potency cannot be simply correlated to Stat activation potency, the quantitative analysis of early signaling is informative. Indeed, in cells with scarce receptors, the two subtypes are equally potent in Jak/Stat activation, while in cells with abundant receptors IFN-α2 is more potent (Fig. ). In vitro measurements of the ligand-receptor interaction have shown large differences in the binding affinity of the two ligands to the receptor chains (see the introduction). To analyze how the binding affinity of the ligand contributes to the differentials observed, we have used two IFN-α2 mutants that exhibit increased affinity to one or the other receptor chain (10
). Thus, we could show that in cells with abundant receptors both IFN-α2 mutants were less potent in Jak/Stat phosphorylation and growth inhibition than natural IFN-α2 (Fig. ), suggesting that the low-affinity ligand is more fit to early signal activation.
In conclusion, even though IFN-α2 presents an apparent binding affinity lower than that of IFN-β (Fig. ), it can possess similar or higher specific bioactivities in cells with abundant receptors. This apparent paradox can be explained if one takes into account also the stability of the ternary complexes formed by IFN-α2 and IFN-β. Indeed, from measuring the rate constants of ternary complex assembly on artificial membranes, the half-life of the ternary complex was found to be considerably higher for IFN-β (100 s) than for IFN-α2 (1 to 5 s) (8
). Thus, IFN-α2 is likely to engage in short-lived ternary complexes and IFN-β in long-lived ternary complexes. These properties are somehow reminiscent of the behavior of the T-cell receptor (TCR) which exhibits high sensitivity and specificity to peptide-major histocompatibility complex ligands (1
). One model proposed to explain the high TCR sensitivity is serial triggering, whereby a ligand of low affinity can sequentially engage multiple TCRs and thus amplify the signal (9
). An activation mode analogous to the one described for the TCR system could be invoked for the type I IFN receptor system. In cells with scarce receptors, engagement of most, if not all, receptors is likely to occur and the half-life of the ternary complex will discriminate between the two IFN subtypes and determine the signaling outcomes. Thus, the long-lived complexes formed with IFN-β will be able to induce a signal or signals which ultimately reinforce apoptosis. In cells with abundant receptors, the number of ternary complexes formed, rather than their half-life, will be critical. In this context, IFN-α2 will be advantaged by sequentially triggering multiple receptors, by virtue of its lower binding activity/affinity. This will amplify Jak/Stat signaling and downstream gene induction (see also the model in Fig. ).
FIG. 9. Model of receptor activation by IFN-α2 versus IFN-β and signaling outcomes The model is based on reported in vitro parameters of binary and ternary complex assembly (see references in text) and on the present cell-based analyses. The left (more ...)
Another system in which two ligands share the same receptor and serial engagement could be invoked is the interleukin-4 (IL-4)/IL-13 system, which signals through the Jak/Stat cascade. On nonhematopoietic cells, both IL-4 and IL-13 bind to a complex made of IL-4 receptor α (IL-4Rα) and IL-13Rα1. The binding affinity of each cytokine for the two chains differs. IL-13 uses as a driver the IL-13Rα1 chain (high affinity) and as a trigger IL-4Rα (low affinity), whereas IL-4 uses IL-4Rα as the driver and IL-13Rα1 as the trigger. Although the complex with IL-13 appears to be the most stable by virtue of a higher affinity of the IL-13/IL-13Rα1 binary complex for IL-4Rα, IL-4 was reported to be more potent than IL-13 at stimulating phosphorylation of Stat6 (15
). This unexpected result may underlie serial triggering, whereby the number of ternary complexes induced by IL-4 is higher than that induced by IL-13 and, as a consequence, IL-4 would activate Stat6 more efficiently.
Here we have also shown that the differential antiproliferative potency of IFN-α2 and IFN-β measured in U5-low/low or in WISH cells is exerted at the level of apoptotic signals and is mediated, at least in part, by differential upregulation of Fas. We can only speculate on the mechanism by which IFN-β promotes higher surface Fas, as this question goes beyond our present study. It has been described that Fas localizes in intracellular stores (3
) and that, upon proapoptotic stimuli, such as IFN-γ, it traffics to the plasma membrane (2
). Therefore, an attractive possibility is that IFN-β somehow affects the intracellular traffic of proapoptotic receptors, such as Fas. In support of this, we found no evidence of Fas
mRNA induction by either IFN subtype (data not shown), suggesting that IFN-β may act posttranscriptionally. Additionally, caspase 9, which is a measure of mitochondrion-dependent apoptosis, was activated to the same extent by the two IFN subtypes (data not shown), suggesting that IFN-β specifically sensitizes cells toward the extrinsic pathway. Overall our data point to a model whereby IFN-β, by assembling tighter complexes, may act upon additional signals, such as phosphatidylinositol 3-kinase/Akt, that somehow affect surface expression of proapoptotic receptors (20
Piehler's group has recently shown that the two IFN subtypes form ternary complexes with similar architectures. Moreover, using fluorescence spectroscopy Piehler's group also showed that the ectodomain of IFNAR1 undergoes a substantial rearrangement of the membrane-distal domains upon IFN binding (27
). Future work will therefore need to address the possibility that in live cells this conformational change represents a signature toward diversification of signals and biological outputs.