Fig. 1. Levels of endogenous and transfected IFNAR1. (A) Surface level of endogenous IFNAR1. The level of IFNAR1 was determined by flow cytometry on the indicated cell lines, using the AA3 mAb. The broken curve represents staining with an isotype matched control mAb. The right panel shows the Tyk2 content in 10 µg of lysate from the indicated cell lines. (B) Surface and total levels of endogenous IFNAR1. WT and 11,1 cells were processed for surface and total IFNAR1 immunoprecipitation (see Materials and methods). The recovered material was separated by SDS–PAGE and analyzed by anti-IFNAR1 immunoblot. The lower panel shows the Tyk2 content in 10 µg of lysate. (C) Surface level of transfected IFNAR1. The level of IFNAR1 was determined by flow cytometry as in (A) on 11,1 cells transiently co-transfected with IFNAR1 and with vector (filled, median fluorescence 11.2), wt Tyk2 (bold, median 48.3) or Δ(1–287) mutant (thin, median 8.6). The broken curve represents IFNAR1 in untransfected 11,1 cells. (D) Surface and total levels of transfected IFNAR1. 11,1 cells were transfected with empty vector (–) or with the plasmids indicated and processed as in (B). (E) Endo H sensitivity of transfected IFNAR1. 11,1 cells were transiently transfected with the plasmids indicated, lysed and subjected to total IFNAR1 immunoprecipitation. Half of the immuno-absorbed material was digested with Endo H and the other half was left undigested. After separation by SDS–PAGE, anti-IFNAR1 immunoblot was performed. The arrow indicates the partly glycosylated form of the receptor (95 kDa) and the arrowhead indicates the Endo H-digested product (64 kDa).

Fig. 3. Tyk2 stabilizes IFNAR1 and IL-10R2 at the plasma membrane. (A) 11,1 cells were co-transfected with human IFNAR1 (R1) and either vector (left) or human (right) Tyk2. Cells were fixed, permeabilized and stained with anti-IFNAR1 and anti-Tyk2 Abs. IFNAR1 staining in Tyk2-positive cells is shown in the right panel. (B) 11,1 cells were co-transfected with the VSV-G epitope tagged murine IFNAR1 (mR1) with either vector (left) or human (right) Tyk2. Staining was with the p5D4 mAb and anti-Tyk2 Abs. IFNAR1 staining in Tyk2-positive cells is shown in the right panel. (C) COS-7 cells were co-transfected with human IFNAR1 (R1) and either vector (left) or human (right) Tyk2. Cells were stained with anti-IFNAR1 and anti-Tyk2 Abs. IFNAR1 staining in Tyk2-positive cells is shown in the right panel. (D) MEF Tyk2–/– cells were co-transfected with human IFNAR1 (R1) and either vector (left) or human (right). Cells were stained with anti-IFNAR1 and anti-Tyk2 Abs. IFNAR1 staining in Tyk2-positive cells is shown in the right panel. (E) 11,1 cells were co-transfected with human IL-10R2 and either vector (left) or human (right). Cells were stained with anti-IL-10R2 and anti-Tyk2 Abs. IL-10R2 staining in Tyk2- positive cells is shown in the right panel. Images were acquired with a CCD camera.

Fig. 5. (A) Schematic representation of mutated forms of IFNAR1. Only the IC region is drawn. The unshaded rectangle in wild-type IFNAR1 (WT) corresponds to the Tyk2 binding region (residues 486–511) (Yan et al., 1996). (B) Localization of mutated forms of IFNAR1. 11,1 cells were co-transfected with the IFNAR1 mutants indicated and either vector (upper panels) or Tyk2 (lower panels). Cells were double stained with anti-IFNAR1 and anti-Tyk2 Abs. Representative IFNAR1 staining profiles in Tyk2-positive cells are shown. (C) Surface expression levels of IFNAR1 mutants. 11,1 cells were co-transfected with the IFNAR1 plasmid indicated and vector (–) or Tyk2 (+) and processed for surface immunoprecipitation as in Figure 1B.

Fig. 7. Basal endocytosis of IFNAR1 is slowed down by Tyk2. COS-7 cells were transfected with IFNAR1 (squares) or with IFNAR1 and Tyk2 (circles). Two days later, cells were harvested, incubated with 5 nM of anti-IFNAR1 [¹²⁵I]64G12 mAb for 1 h at 4°C and then warmed to 37°C to allow endocytosis to proceed. At the times indicated, the internalized material was measured as the cell-associated radioactivity remaining after the acid pH wash (see Materials and methods). Background values obtained from cells transfected with only empty vector were substracted. The plot shows the ratios of internalized (acid-resistant) to surface radioactivity. The inset illustrates the percentage of intracellular (empty bars) and surface (filled bars) radioactivity relative to total radioactivity after 90 min of incubation at 37°C for cells transfected with IFNAR1 (R1) and cells co-transfected with IFNAR1 and Tyk2 (R1+Tyk2). This figure is representative of one of three experiments yielding comparable results.

Fig. 2. In the absence of Tyk2 IFNAR1 accumulates in an endosomal compartment. (A) 11,1 cells were transfected with human IFNAR1 (R1) and 24 h later they were incubated with Alexa⁴⁸⁸-coupled transferrin for 60 min at 37°C. Cells were fixed, permeabilized and stained with anti-IFNAR1 mAbs, followed by Texas Red-coupled secondary Abs, and analyzed by confocal microscopy as described in Materials and methods. IFNAR1, red; transferrin, green; merging of the two signals is shown in the overlay. (B) IFNAR1-transfected cells were fixed, permeabilized and stained with anti-IFNAR1 and anti-EEA1 Abs, followed by Texas Red- and Alexa⁴⁸⁸-coupled secondary Abs. IFNAR1, red; EEA1, green. White arrows in the overlay indicate some of the vesicles where IFNAR1-positive and EEA1-positive staining overlapped. (C) IFNAR1-transfected cells were fixed, permeabilized and stained with anti-IFNAR1 and anti-Lamp1 Abs, followed by Texas Red- and Alexa⁴⁸⁸-coupled secondary Abs. IFNAR1, red; Lamp1, green. (D) 11,1 cells were co-transfected with IFNAR1 and the pECFP-Golgi marker and allowed to internalize the anti-IFNAR1 AA3 mAbs for 60 min at 37°C. Cells were fixed, permeabilized and stained with Texas Red-coupled secondary Abs. IFNAR1, red; CFP fluorescence, green. (E) IFNAR1-transfected cells were allowed to internalize both Alexa⁴⁸⁸-coupled transferrin and anti-IFNAR1 AA3 mAbs for 60 min at 37°C. Cells were processed as in (D). IFNAR1, red; transferrin, green. The white arrow in the overlay indicates one of several vesicles stained by both the internalized mAbs and transferrin. Scale bar, 5 µm.

Fig. 4. Localization of IFNAR1 co-expressed with Tyk2 mutants. (A) 11,1 cells co-transfected with IFNAR1 (R1) and the Tyk2 constructs indicated [schematized in (B)] were stained with anti-IFNAR1 and anti-Tyk2 Abs, as in Figure 3A. Representative IFNAR1 staining profiles in Tyk2-positive cells are shown. Images were acquired with a CCD camera. (B) Schematic representation of the domain organization of Tyk2 and of the various constructs that were tested for their effect on IFNAR1 localization. The N region (590 aa) includes the FERM and the SH2-like domain. The bar over the FERM domain points to the minimal IFNAR1 binding domain (Richter et al., 1998). K930R is a kinase-dead form with a Lys-to-Arg substitution mutation in the ATP-binding site (Gauzzi et al., 1996). KL, kinase-like; TK, tyrosine kinase.

Fig. 6. (A) Tyk2 delays IFNAR1 degradation. 11,1 cells were co-transfected with IFNAR1 (R1) and the empty vector or the Tyk2 constructs indicated. The following day, CHX was added for the periods indicated. Total lysates were resolved by SDS–PAGE and blotted with anti-IFNAR1 Abs (top) or anti-Tyk2 Abs (bottom). (B) Pharmacological inhibition of IFNAR1 degradation. 11,1 cells were transfected with IFNAR1 and the following day cells were left untreated or treated for 2 h with CHX alone or in combination with the inhibitor indicated. Total lysates were resolved by SDS–PAGE and blotted with anti-IFNAR1 Abs. (C) Stability of IFNAR1 mutants in CHX-treated cells. Wild-type and three truncated forms of IFNAR1 (scheme in Figure 5A) were transfected and analyzed as in (A). The WT form was also analyzed when co-transfected with Tyk2. The densitometric scan of the signal is shown. For each construct, the total receptor level in cells not treated with the drug was taken as 100%. The data are from one of three experiments yielding comparable results.