Basal ubiquitin-independent internalization of interferon alpha receptor is prevented by Tyk2-mediated masking of a linear endocytic motif - PubMed (original) (raw)

Basal ubiquitin-independent internalization of interferon alpha receptor is prevented by Tyk2-mediated masking of a linear endocytic motif

K G Suresh Kumar et al. J Biol Chem. 2008.

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 IFNalpha 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 Tyr-based 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.

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Figures

FIGURE 1.

FIGURE 1.

Sequence alignment of the proximal portions of the intracellular domain of IFNAR1 receptors from different species. All currently known sequences of IFNAR1 are depicted starting from the end of the transmembrane domain. Putative endocytic motifs are depicted in bold letters. In human IFNAR1, the position of Tyr-466 is noted by number, and the amino acid residues situated within a region shown to participate in the interaction with Tyk2 (28) are_underlined_.

FIGURE 2.

FIGURE 2.

Knockdown of Tyk2 decreases the levels of endogenous IFNAR1 in 293T cells. A, characterization of shRNA constructs against Tyk2. 293T cells were transfected with the indicated constructs and the levels of Tyk2 and α-tubulin (α_-Tub_) were analyzed by immunoblotting using respective antibodies. Mock, mock-transfected; shCON represents cells transfected with shRNA against green fluorescent protein.B, effect of either Tyk2 or Jak2 knockdown on the steady-state level of endogenous IFNAR1 analyzed by immunoprecipitation with anti-IFNAR1 antibody EA12 followed by immunoblotting with anti-IFNAR1 antibody GB8.Asterisks denote nonspecific bands that reflect equal protein loading. Levels of Tyk2 and Jak2 in whole cell extracts analyzed by immunoblotting using appropriate antibodies are also shown. C, effect of either Tyk2 (closed squares) or Jak2 (closed circles) knockdown on the internalization of endogenous IFNAR1 in 293T cells that were not treated with the ligand measured using the anti-IFNAR1 AA3 antibody-based fluorescent assay as described in Ref. and outlined under “Experimental Procedures.” An average result of four experiments (±S.E.) is shown.

FIGURE 3.

FIGURE 3.

Knockdown of Tyk2 promotes basal internalization of IFNAR1 in an ubiquitination-independent manner. A, internalization of endogenous IFNAR1 in 293T cells transfected with indicated shRNA constructs (circles, control shRNA, and squares, shRNA against Tyk2) and treated (closed symbols) or not (open symbols) with IFNα was analyzed using the anti-IFNAR1 AA3 antibody-based fluorescent assay as described in Ref. and outlined under “Experimental Procedures.” shCON represents cells transfected with shRNA against green fluorescent protein.B, in vivo ubiquitination of FLAG-tagged IFNAR1 proteins (wild type (WT) or SA mutant, as indicated) expressed in 293T cells was assessed by denaturing immunoprecipitation using anti-FLAG (M2) antibody followed by immunoblotting (IB) using either anti-ubiquitin (Ub, upper panel) or anti-FLAG (lower panel) antibodies. C, internalization of FLAG-tagged IFNAR1 proteins (open symbols, wild type, or closed symbols, ubiquitination-deficient SA mutant) in 293T cells transfected with control shRNA (circles) or shRNA against Tyk2 (squares) in the absence of IFN.

FIGURE 4.

FIGURE 4.

Tyk2 prevents basal internalization of IFNAR1 in a manner that depends on Tyk2-IFNAR1 interaction and on integrity of the Tyr-based endocytic motif. A, interaction of wild type Tyk2 and the Y103A/F104A double mutant (Tyk2YFAA) with FLAG-IFNAR1 co-expressed in 293T cells analyzed by immunoprecipitation (IP) and immunoblotting (IB) with the indicated antibodies. Loading of IFNAR1 into the final reaction was normalized to achieve comparable levels of this FLAG-tagged protein in all lanes. B, effect of coexpression of wild type Tyk2 or the Y103A/F104A double mutant (Tyk2YFAA) on surface (S) and total (T) levels of IFNAR1 in Tyk2-null 11,1 cells. Cell surface IFNAR1 was collected by streptavidin bead-based pull-down from the lysates of cells that underwent surface biotinylation (as described under “Experimental Procedures”). Both surface and total levels were analyzed by immunoblotting using anti-FLAG M2 antibody. Levels of Tyk2 and β-actin in whole cell lysates were also analyzed by immunoblotting.C, internalization of FLAG-tagged IFNAR1 co-expressed with empty vector (Vec, closed squares), wild type Tyk2 (open squares), or the Y103A/F104A double mutant (Tyk2YFAA) (closed triangles) in 11,1 cells was determined as in Fig. 3_C_. D, internalization of the IFNAR1 mutants (IFNAR1S535A,squares; IFNAR1 S535A/Y466F, triangles) co-expressed with (open symbols), or without (closed symbols) Tyk2 was analyzed as in panel C.

FIGURE 5.

FIGURE 5.

Proximity of the Tyk2 binding site to the Tyr-based linear endocytic motif within IFNAR1 is required for Tyk2-mediated inhibition of basal endocytosis of IFNAR1. A, graphic depiction of IFNAR1 mutants. The Tyr-based endocytic motif is denoted. WT, wild type; BD, binding domain; SP, spacer. B, interaction of IFNAR1 mutants with Tyk2 was analyzed by immunoprecipitation and immunoblotting as described in the legend for Fig. 4.C, internalization of wild type IFNAR1 (closed squares), spacer mutant (SP, closed triangles), and spacer + Y466F mutant (SPYF, open diamonds) in 293T cells in the absence of IFN was analyzed as described in the legend for Fig. 3.

FIGURE 6.

FIGURE 6.

Interaction of Tyk2 with IFNAR1 physically shields the receptor from binding AP50. A, interaction of endogenous IFNAR1 with endogenous AP50 (μ2) was measured in 11,1 Tyk2-null cells or in their KR derivatives (stably expressing catalytically inactive Tyk2). Cells were pretreated with methylamine (40 m

m

, 2 h), an inhibitor of the lysosomal pathway, to prevent IFNAR1 degradation. The interaction was assessed by immunoprecipitation (IP) using anti-IFNAR1 (R1) antibodies followed by immunoblotting using the indicated antibodies. Levels of the AP50 subunit and Tyk2 in whole cell lysates (WCE) are also shown. B, transfection of increasing doses of HA-tagged Tyk2 inhibits the interaction between FLAG-tagged IFNAR1 and HA-tagged AP50 in 293T cells. This interaction was assessed by immunoprecipitation using anti-FLAG antibody followed by immunoblotting (IB) using indicated antibodies. The amount of immunoprecipitated IFNAR1 was normalized (upper panel) to achieve comparable levels of loading. Levels of FLAG-IFNAR1, HA-Tyk2, and HA-AP50 in whole cell lysates are also shown. C, effects of expression of either wild type Tyk2 or the Y103A/F104A double mutant (Tyk2YFAA) on the IFNAR1-AP50 interaction assessed as described in panel B.Mock, mock-transfected.

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