Basal Ubiquitin-independent Internalization of Interferon α Receptor Is Prevented by Tyk2-mediated Masking of a Linear Endocytic Motif

Abstract

Linear endocytic motifs of signaling receptors as well as their ubiquitination determine the rate of ligand-induced endocytosis that mediates down-regulation of these receptors and restricts the magnitude and duration of their respective signal transduction pathways. We previously hypothesized that, in the absence of its cognate ligand, type I interferon (IFN), the IFN␣ receptor chain 1 (IFNAR1) receptor chain is protected from basal endocytosis by a hypothetical masking complex that prevents the Tyr-based endocytic motif within IFNAR1 from interacting with components of the adaptin protein complex 2 (AP2). Here we identify a member of the Janus kinase (Jak) family, Tyk2, as a component of such a masking complex. In the absence of ligand or of receptor chain ubiquitination, binding of Janus kinase Tyk2 within the proximity of the Tyr-based linear motif of IFNAR1 is required to prevent IFNAR1 internalization and to maintain its cell surface expression. Furthermore, interaction experiments revealed that Tyk2 physically shields this Tyrbased motif from the recognition by the AP50 subunit of AP2. These data delineate a long-sought ligand-and ubiquitin-independent mechanism by which Tyk2 contributes to both the regulation of total IFNAR1 levels as well as the regulation of the cell surface density of this receptor chain.Cells react to diverse environmental stimuli by expressing specific receptors that recognize these stimuli and initiate specific signaling pathways that enable a cell or a tissue to cope with an altered environment. Down-regulation of these signaling receptors represents the most specific mode of limiting the magnitude and duration of given signal transduction pathways. For transmembrane receptors displayed at the cell surface, ligand-stimulated endocytosis is a major mechanism by which the ability of a cell to react to a ligand is restricted. In addition, basal ligand-independent internalization may determine how responsive a naive cell could be to a subsequent encounter with a particular ligand.Mechanisms mediating internalization of signaling receptors involve a dynamic plasma membrane exchange resulting in bulk endocytosis and a cargo-specific clathrin-dependent endocytosis. Interaction of clathrin lattices formed on the plasma membrane with cargo receptors relies on adaptor complexes such as the adaptin protein-2 complex (AP2), 4 which recognizes specific linear endocytic motifs present within the cytoplasmic domains of the target receptor. For example, the 2 subunit of AP2 (also termed AP50) is known to recognize Tyr-based linear endocytic motifs and to enable the AP2-dependent tethering of cargo to clathrin molecules (reviewed in Refs. 1-4).Receptor ubiquitination has also emerged as a key endocytic signal for numerous eukaryotic cell surface receptors (reviewed in Refs. 5-8). For several receptors (such as epidermal growth factor receptor, growth hormone receptor, interferon ␣ receptor chain 1 (IFNAR1)), ubiquitination is stimulated by the ligand. This stimulation is mediated by the ligand-ind...

Palmitoylation of Interferon-α (IFN-α) Receptor Subunit IFNAR1 Is Required for the Activation of Stat1 and Stat2 by IFN-α

“…We first tested the hypothesis of a defect in kinase activation. Tyk2 was a likely candidate because it is constitutively associated with IFNAR1 on a stretch of amino acids close to the palmitoylated cysteine (40), and it has been recently shown that the accessibility of Tyk2 to IFNAR1 depends on post-translational modifications of IFNAR1 (48). We probed this interaction using BRET, which is more sensitive than classical immunoprecipitation assays.…”

Important scaffold function of the Janus kinase 2 uncovered by a novel mouse model harboring a Jak2 activation-loop mutation

“…However, there might be subtle differences for the expression in IFNGR between mice and humans that have to be addressed in the future. Interestingly, for Tyk2-null human cells a lack of cell surface expression of the IFN-a receptor 37 due to unmasking of an endocytic motif that is missing in the mouse receptor has been reported. 38 To prove that our mouse model is a suitable model for the investigation of kinase-independent Jak2 functions, we further investigated the role of Jak2-K882E.…”

Hemagglutinin of Influenza A Virus Antagonizes Type I Interferon (IFN) Responses by Inducing Degradation of Type I IFN Receptor 1

“…Mammalian expression plasmids encoding C-terminal 3ϫFLAG-tagged wild-type (WT) IFNAR1 and its phosphorylation-incompetent mutant (IFNAR1-S535/539A) were previously used (22,42,43). Mammalian expression plasmids encoding 8ϫHA-tagged WT ubiquitin (tHA-Ub) and mutants (tHA-Ub-K48 and tHA-Ub-K63) were provided by Mark Hannink (University of Missouri-Columbia) (44) (45)(46)(47).…”