Type I Interferon Signaling Is Decoupled from Specific Receptor Orientation through Lenient Requirements of the Transmembrane Domain

Abstract

Type I interferons serve as the first line of defense against pathogen invasion. Binding of IFNs to its receptors, IFNAR1 and IFNAR2, is leading to activation of the IFN response. To determine whether structural perturbations observed during binding are propagated to the cytoplasmic domain, multiple mutations were introduced into the transmembrane helix and its surroundings. Insertion of one to five alanine residues near either the N or C terminus of the transmembrane domain (TMD) likely promotes a rotation of 100°and a translation of 1.5 Å per added residue. Surprisingly, the added alanines had little effect on the binding affinity of IFN to the cell surface receptors, STAT phosphorylation, or gene induction. Similarly, substitution of the juxtamembrane residues of the TMD with alanines, or replacement of the TMD of IFNAR1 with that of IFNAR2, did not affect IFN binding or activity. Finally, only the addition of 10 serine residues (but not 2 or 4) between the extracellular domain of IFNAR1 and the TMD had some effect on signaling. Bioinformatic analysis shows a correlation between high sequence conservation of TMDs of cytokine receptors and the ability to transmit structural signals. Sequence conservation near the TMD of IFNAR1 is low, suggesting limited functional importance for this region. Our results suggest that IFN binding to the extracellular domains of IFNAR1 and IFNAR2 promotes proximity between the intracellular domains and that differential signaling is a function of duration of activation and affinity of binding rather than specific conformational changes transmitted from the outside to the inside of the cell.Type I interferons (IFNs) orchestrate the antiviral innate immunity in vertebrates. They consist of 16 members in humans as follows: 12 different IFN␣ subtypes, as well as IFN␤, IFN, IFN⑀, and IFN. They are clustered on the short arm of chromosome 9. All type I interferons elicit their innate and adaptive immune responses after binding to the receptor and forming the IFNAR1-IFN-IFNAR2 ternary complex (1, 2), the formation of which is essential for signaling. Both IFNAR1 and IFNAR2 belong to the class II helical cytokine receptors with four fibronectin type III-like subdomains for IFNAR1 and two subdomains for IFNAR2. The extracellular domains are followed by a single helix of 21 amino acids spanning the membrane, which in turn is connected to mostly natively unstructured intracellular domains and their associated effectors (Fig. 1A) (3). Cells lacking one of the receptors lack normal IFN signaling (4, 5). Structurally different members of type I IFNs bind to the same cell surface receptor but mediate differential responses that result from differences in binding affinities, concentration of IFN, and duration of activation (5-13). Differential signaling is realized through robust (antiviral) versus tunable (antiproliferative and immunomodulatory) gene induction leading to different phenotypic outcomes (14,15). Receptor dimerization drives the activation of cytosolic associated Janus family kina...

A hot spot on interferon α/β receptor subunit 1 (IFNAR1) underpins its interaction with interferon-β and dictates signaling

“…Mechanistically, these results may, therefore, point to a role for the identified IFN-␤-IFNAR1-SD3 interface in differentially regulating or mediating alternative IFN-␤-IFNAR1-driven signaling events or pathways. Our data, demonstrating the functional importance of residues at the IFN-␤-IFNAR1-SD3 interface, are in contrast to alanine mutations introduced to the (juxta) transmembrane region which predictably had no effect on IFN binding affinity or signaling (28).…”

Dynamic Modulation of Binding Affinity as a Mechanism for Regulating Interferon Signaling

“…As shown by us previously, it is unlikely that the movements of the EC domain will affect the arrangement of the IC domain (Sharma et al , 2016). However, such movements may be essential to mediating ligand specificity and binding affinity, and may affect signaling functions.…”

“…IFNs induce a cellular response upon forming a ternary complex with two surface receptors, IFNAR1 and IFNAR2 (Thomas et al , 2011). Binding of IFN to the extracellular (EC) domains of these receptors results in bringing their intracellular (IC) domains into close proximity, upon which their associated tyrosine kinases (JAKs) phosphorylate IC receptor chains and associated effector proteins (Stark and Darnell, 2012; Sharma et al , 2016). …”

“…Recently we have shown that IFN activation does not require specific conformational changes transmitted from the outside to the inside of the cell, suggesting that JAK/STAT activation is a function of the ligand-driven proximity and the duration of engagement (Sharma et al , 2016). The family of IFNs comprises sixteen members in humans (Uze et al , 2007), which are not all functionally redundant (Brierley and Fish, 2002; Foster et al , 2004; Severa et al , 2006; Jaks et al , 2007; Marijanovic et al , 2007; Kalinke and Prinz, 2012).…”

“…Both global and local conformational changes were observed for IFNα2 and IFNβ. While the conformational changes at the EC region of IFNAR1 may be important, IFN signaling remains intact when the TM helix connecting the EC and IC IFNAR1 domains was mutated in any possible way (Sharma et al , 2016), presumably due to the unstructured segments flanking the TM domain on both sides.…”