Site-specific ubiquitination exposes a linear motif to promote interferon-α receptor endocytosis

Abstract

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCFβTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.

Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway

“…For purification of the ubiquitinated species of PRLr, 50 10-cm-diameter dishes of 293T cells were used for transfection and coexpression of WT ubiquitin and Flag-tagged PRLr or a vector control by the calcium phosphate method. Purification of ubiquitinated PRLr was done similarly to purification of ubiquitinated IFNAR1 as previously described (24). Briefly, cells were treated with PRL (50 ng/ml for 30 min) and lysed using NP-40 lysis buffer containing 200 mM NaCl, protease inhibitors, and 10 mM NEM (N-ethyl maleimide).…”

“…Reversible cell surface biotinylation. Cell surface biotinylation was performed as previously described (20,24,39). This procedure uses immunoblotting to measure levels of biotinylated proteins that are protected from debiotinylation as a result of being internalized.…”

“…pH v measurement. To monitor the endocytic trafficking of internalized PRLr, the vesicular pH (pH v ) of cargo-containing vesicles was determined by fluorescence ratio image analysis (FRIA) essentially as described for CD4 or IFNAR1 (3,24). Cell surface PRLr were labeled with anti-HA primary antibody (Covance MMS101R) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary Fab (Jackson ImmunoResearch Laboratories, West Grove, PA) by incubating primary (1/500 dilution) and secondary (1/500 dilution) antibodies together routinely for 1 h at 37°C in the presence of 50 ng of PRL (WT or antagonist)/ml.…”

“…Ubiquitinated cargo destined for lysosomal degradation generally remains associated with the endosome as the vesicles mature and decrease their pH, progressing to vesicles of the late endosome and lysosome (11,41). While any type of polyubiquitination appeared to suffice for the lysosomal targeting of IFNAR1 (24), the role of specific types of ubiquitination for sorting of other signaling receptors is yet to be determined.…”

“…We have recently demonstrated that linkages of both K48 and K63 types were required for efficient internalization of the alpha/beta interferon receptor chain IFNAR1 (24). We pro-posed that interaction of specific ubiquitin moieties with diverse linear endocytic motifs within individual receptors is a major mechanism that regulates the rate of receptor endocytosis.…”