Alpha and beta interferons (IFN-␣Type I interferons (IFNs) form a family of multifunctional cytokines initially described for their direct antiviral effect but now also recognized as major elements of the immune response (19,46). Differential activities of IFN subtypes have been reported (3) and used in the clinic for the treatment of various pathologies, including viral hepatitis (IFN-␣2) and multiple sclerosis (IFN-␤) (32). All type I IFNs are recognized by a single shared receptor composed of two transmembrane proteins, IFNAR1 and IFNAR2. Because of the much faster k on and much slower k off of IFN-␣2 towards IFNAR2 than those measured for IFNAR1 (18,34,40), a two-step assembling mechanism was proposed for the interaction between IFN and the two receptors (Fig. 1B). After binding of IFN-␣2 to IFNAR2 (k a1 ), IFNAR1 transiently associates in a second step to the complex (k a2 ) (18,25). Owing to the short lifetime of the IFN-␣2-IFNAR1 interaction, the complex dissociates (k d2 ) and reassociates (k a2 ) in a fast manner. Thus, depending on the receptor surface concentrations and ligand binding affinities, only part of the bound ligand is involved in the active ternary complex.After formation of the ternary complex, the interferon signal is transduced through the receptor-associated JAK kinases, with the STAT transcription factors as their main targets (3).

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Ligand-receptor interactions within the plane of the plasma membrane play a pivotal role for transmembrane signaling. The biophysical principles of protein-protein interactions on lipid bilayers, though, have hardly been experimentally addressed. We have dissected the interactions involved in ternary complex formation by ligand-induced cross-linking of the subunits of the type I interferon (IFN) receptors ifnar1 and ifnar2 in vitro. The extracellular domains ifnar1-ectodomain (EC) and ifnar2-EC were tethered in an oriented manner on solid-supported lipid bilayers. The interactions of IFNa2 and several mutants, which exhibit different association and dissociation rate constants toward ifnar1-EC and ifnar2-EC, were monitored by simultaneous label-free detection and surface-sensitive fluorescence spectroscopy. Surface dissociation rate constants were determined by measuring ligand exchange kinetics, and by measuring receptor exchange on the surface by fluorescence resonance energy transfer. Strikingly, approximately three-times lower dissociation rate constants were observed for both receptor subunits compared to the dissociation in solution. Based on these directly determined surface-dissociation rate constants, the surface-association rate constants were assessed by probing ligand dissociation at different relative surface concentrations of the receptor subunits. In contrast to the interaction in solution, the association rate constants depended on the orientation of the receptor components. Furthermore, the large differences in association kinetics observed in solution were not detectable on the surface. Based on these results, the key roles of orientation and lateral diffusion on the kinetics of protein interactions in plane of the membrane are discussed.

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